Apoptin-associating protein

ABSTRACT

The invention relates to the field of apoptosis. The invention provides novel therapeutic possibilities, for example, novel combinatorial therapies or novel therapeutic compounds that can work alone, sequentially to, or jointly with Apoptin, especially in those cases wherein p53 is (partly) nonfunctional.

[0001] Priority Claim: This application claims priority under 35 U.S.C. §119 of European Patent applications EP 00250118.7, filed on Apr. 7, 2000 and EP 00200169.1, filed on Jan. 17, 2000, the contents of both of which are incorporated by this reference including all figures, claims and sequences identified therein.

BACKGROUND OF THE INVENTION

[0002] The invention relates to the field of apoptosis. Apoptosis is an active and programmed physiological process for eliminating superfluous, altered or malignant cells (Earnshaw, 1995; Duke et al., 1996). Apoptosis is characterized by shrinkage of cells, segmentation of the nucleus, condensation and cleavage of DNA into domain-sized fragments in most cells followed by internucleosomal degradation. The apoptotic cells fragment into membrane-enclosed apoptotic bodies. Finally, neighboring cells and/or macrophages will rapidly phagocytose these dying cells (Wylie et al., 1980; White, 1996). Cells grown under tissue-culture conditions and cells from tissue material can be analyzed for being apoptotic with agents staining DNA, for example, DAPI, which stains normal DNA strongly and regularly, whereas apoptotic DNA is stained weakly and/or irregularly (Noteborn et al., 1994; Telford et al., 1992).

[0003] The apoptotic process can be initiated by a variety of regulatory stimuli (Wyllie, 1995; White, 1996; Levine, 1997). Changes in the cell survival rate play an important role in human pathogenesis of diseases, for example, in cancer development and autoimmune diseases, where enhanced proliferation or decreased cell death (Kerr et al., 1994; Paulovich, 1997) is observed. A variety of chemotherapeutic compounds and radiation have been demonstrated to induce apoptosis in tumor cells, in many instances via wild-type p53 protein (Thompson, 1995; Bellamy et al., 1995; Steller, 1995; McDonell et al., 1995).

[0004] Many tumors, however, acquire a mutation in p53 during their development, often correlating with poor response to cancer therapy. Certain transforming genes of tumorigenic DNA viruses can inactivate p53 by directly binding to it (Teodoro, 1997). An example of such an agent is the large T antigen of the tumor DNA virus SV40. For several (leukemic) tumors, a high expression level of the proto-oncogene Bcl-2 or Bcr-abl is associated with a strong resistance to various apoptosis-inducing chemotherapeutic agents (Hockenberry 1994; Sachs and Lotem, 1997).

[0005] For such tumors lacking functional p53 (representing more than half of the tumors), alternative antitumor therapies are under development based on induction of apoptosis independent of p53 (Thompson 1995; Paulovich et al., 1997). One has to search for the factors involved in induction of apoptosis, which do not need p53 and/or can not be blocked by antiapoptotic activities, such as Bcl-2 or Bcr-abl-like ones. These factors might be part of a distinct apoptosis pathway or might be (far) downstream of the apoptosis inhibiting compounds.

[0006] Apoptin is a small protein derived from chicken anemia virus (CAV; Noteborn and De Boer, 1995; Noteborn et al., 1991; Noteborn et al., 1994, 1998a) which can induce apoptosis in human malignant and transformed cell lines, but not in untransformed human cell cultures. In vitro, Apoptin fails to induce programmed cell death in normal lymphoid, dermal, epidermal, endothelial and smooth-muscle cells. However, when normal cells are transformed they become susceptible to apoptosis by Apoptin. Long-term expression of Apoptin in normal human fibroblasts revealed that Apoptin has no toxic or transforming activity in these cells (Danen-van Oorschot, 1997 and Noteborn, 1996).

[0007] In normal cells, Apoptin was found predominantly in the cytoplasm, whereas in transformed or malignant cells, i.e., characterized by hyperplasia, metaplasia or dysplasia, it was located in the nucleus, suggesting that the localization of Apoptin is related to its activity (Danen-van Oorschot et al. 1997).

[0008] Apoptin-induced apoptosis occurs in the absence of functional p53 (Zhuang et al., 1995a) and cannot be blocked by Bcl-2, Bcr-abl (Zhuang et al., 1995), or the Bcl-2-associating protein BAG-1 (Danen-Van Oorschot, 1997a; Noteborn, 1996).

[0009] Therefore, Apoptin is a therapeutic compound for the selective destruction of tumor cells or other hyperplasia, metaplasia or dysplasia, especially for those tumor cells which have become resistant to (chemo)-therapeutic induction of apoptosis due to the lack of functional p53 and (over)-expression of Bcl-2 and other apoptosis-inhibiting agents (Noteborn and Pietersen, 1998). It appears, that even premalignant, minimally transformed cells are sensitive to the death-inducing effect of Apoptin. In addition, Noteborn and Zhang (1998) have shown that Apoptin-induced apoptosis can be used to diagnose cancer-prone cells and to treat cancer-prone cells.

[0010] The fact that Apoptin does not induce apoptosis in normal human cells, at least not in vitro, shows that a toxic effect of Apoptin treatment in vivo will be very low. Noteborn and Pietersen (1998) and Pietersen et al. (1999) have provided evidence that adenovirus expressed Apoptin does not have an acute toxic effect in vivo. In addition, in nude mice it was shown that Apoptin has a strong antitumor activity.

[0011] However, to further enlarge the array of therapeutic anticancer or antiautoimmune disease compounds available in the art, additional therapeutic compounds are desired that are designed to work alone, sequentially to, or jointly with Apoptin, especially in those cases wherein p53 is (partly) nonfunctional.

DISCLOSURE OF THE INVENTION

[0012] The invention provides novel therapeutic possibilities, for example, novel combinatorial therapies or novel therapeutic compounds that can work alone, sequentially to, or jointly with Apoptin, especially in those cases wherein p53 is (partly) nonfunctional.

[0013] In a first embodiment, the invention provides an isolated or recombinant nucleic acid or functional equivalent or fragment thereof encoding an Apoptin-associating proteinaceous substance capable of providing apoptosis, alone or in combination with other apoptosis inducing substances, such as Apoptin. Proteinaceous substance is herein defined as a substance comprising a peptide, polypeptide or protein, optionally having been modified by, for example, glycosylation, myristilation, phosphorylation, the addition of lipids, by homologous or heterologous di- or multimerisation, or any other (posttranslational) modifications known in the art.

[0014] Apoptin-associating is herein defined as belonging to the cascade of substances specifically involved in the cascade of events found in the apoptosis pathway as inducable by Apoptin, preferably those substances that are specifically involved in the p53-independent apoptosis pathway.

[0015] In a preferred embodiment, the invention provides an isolated or recombinant nucleic acid or functional equivalent or fragment thereof encoding an Apoptin-associating proteinaceous substance capable of providing apoptosis derived from a cDNA library, preferably a vertebrate cDNA library, such as derivable from poultry, but more preferably a mammalian cDNA library, preferably wherein said cDNA library comprises human cDNA An Apoptin-associating proteinaceous substance obtained by determining a vertebrate analogue (preferably human) of an Apoptin-associating proteinaceous substance derived from an invertebrate cDNA library is also included.

[0016] In another embodiment, the invention provides an isolated or recombinant nucleic acid or functional equivalent or fragment thereof encoding an Apoptin-associating proteinaceous substance capable of providing apoptosis capable of hybridizing to a nucleic acid molecule encoding an Apoptin-associating proteinaceous substance capable of providing apoptosis as shown in FIGS. 1, 2, 5 and/or 7, in particular encoding a novel protein capable of providing apoptosis or functional equivalent or functional fragment thereof called Apoptin-associating protein 2 or 3, abbreviated herein also as AAP-2 or AAP-3. FIG. 1 and 2 show an approximately 1100 and 900 bp fragment of the AAP-2 fragment as depicted in FIG. 7. All 3 nucleotide sequences encode a protein with at least the capability of binding to Apoptin and providing apoptosis. Of course, an isolated or recombinant nucleic acid or functional equivalent or fragment thereof encoding an additional Apoptin-associating proteinaceous substance capable of associating with the AAP-2 or AAP-3 protein are herewith also provided, means and methods to arrive at such an additional protein located in the Apoptin cascade follow those of the detailed description given herein. Knowledge derived from studying the AAP-2 and/or AAP-3 clones is exploited to determine a functional pathway in which AAP-2 and/or AAP-3 is/are involved, thus allowing the design of a therapeutic means of intervening in such a pathway.

[0017] A functional equivalent or a functional fragment thereof is a derivative or a fragment having the same kind of activity possibly in different amounts. It is clear to a person skilled in the art that there are different ways of arriving at a functional equivalent or functional fragment. A functional equivalent can, for example, be a point mutant or a deletion mutant or a equivalent derived from another species. Another way to arrive at a functional equivalent is a molecular evolution of equivalents and/or fragments having the same kind of activity possibly in different amounts.

[0018] In particular, the invention provides an isolated or recombinant nucleic acid or functional equivalent or fragment thereof encoding an Apoptin-associating proteinaceous substance capable of providing apoptosis being at least 60% homologous, preferably at least 70%, more preferably at least 80%, even more preferably 90% and most preferably at least 95% homologous to a nucleic acid molecule, or to a functional equivalent or functional fragment thereof, encoding an Apoptin-associating proteinaceous substance as shown in FIGS. 1, 2, 5 or 7.

[0019] Furthermore, the invention provides a vector comprising a nucleic acid according to the invention. Examples of such a vector are given in the detailed description given herein, such as vector pMT2SM-AAP-2 or -AAP-3, pMT2SM vector expressing Myc-tagged AAP-2 or AAP-3 cDNA, a plasmid expressing an Apoptin-associating protein fragment, and so on. These and other vectors are, for example, useful in finding additional Apoptin-associating proteinaceous substances from the cascade, as defined above.

[0020] In yet another embodiment, the invention provides a vector comprising a nucleic acid according to the invention, said vector comprising a gene-delivery vehicle, making the invention very useful in gene therapy. By equipping a gene delivery vehicle with a nucleic acid according to the invention, and by targeting said vehicle to a cell or cells that have been over-proliferating and/or have shown decreased death rates, said gene delivery vehicle provides said cell or cells with the necessary means for apoptosis, providing far reaching therapeutic possibilities.

[0021] Furthermore, the invention provides a host cell comprising a nucleic acid or a vector according to the invention. Examples comprise transformed or transfected bacterial or yeast cells as described in the detailed description herein. Preferred is a host cell according to the invention which is a transformed eukaryotic cell such as a yeast cell or a vertebrate cell, such as mammalian or Cos cells transformed or transfected with a nucleic acid or vector according to the invention. Said cells are in general capable to express or produce a proteinaceous substance capable of providing apoptosis with the ability to associate with Apoptin.

[0022] The invention furthermore provides an isolated or recombinant Apoptin-associating proteinaceous substance capable of providing apoptosis. As, for example, shown herein in FIG. 4, expression of such Apoptin-associating proteinaceous substance in cells such as tumor cells or other over-proliferating cells, induces the apoptic process. It can do so alone or in the presence of other apoptosis inducing substances such as Apoptin and especially so independent of p53 showing that also in those cases where (functional) p53 is absent, apoptosis can be induced by a substance according to the invention. In particular, the invention provides a proteinaceous substance according to the invention encoded by a nucleic acid, for example, comprising at least a part of an amino acid sequence as shown in FIG. 4 or a functional equivalent or functional fragment thereof capable of providing apoptosis alone or in combination with other apoptosis inducing substances such as Apoptin.

[0023] The invention also provides an isolated or synthetic antibody specifically recognising a proteinaceous substance or functional equivalent or functional fragment thereof according to the invention. Examples of such an antibody are given in the detailed description continued herein. Such an antibody is, for example, obtainable by immunizing an experimental animal with an Apoptin-associating proteinaceous substance or an immunogenic fragment or equivalent thereof and harvesting polyclonal antibodies from said immunized animal (as shown herein in the detailed description) or obtainable by other methods known in the art such as by producing monoclonal antibodies or (single chain) antibodies or binding proteins expressed from recombinant nucleic acid derived from a nucleic acid library, for example, obtainable via phage display techniques.

[0024] With such an antibody, the invention also provides a proteinaceous substance specifically recognizable by such an antibody according to the invention, for example, obtainable via immunoprecipitation, Western Blotting, or other immunological techniques known in the art.

[0025] Furthermore, the invention provides use of a nucleic acid, vector, host cell, or proteinaceous substance according to the invention for the induction of tumor-specific apoptosis, as, for example, shown in FIG. 4. In particular, such use is provided wherein said apoptosis is p53-independent. In particular, such use is also provided further comprising use of a nucleic acid encoding Apoptin or a functional equivalent or fragment thereof or use of Apoptin or a functional equivalent or fragment thereof As can be seen from FIG. 4, combining these Apoptin-inducing substances increases the apoptosis percentage of treated tumor cells.

[0026] Such use as provided by the invention is particularly useful from a therapeutic viewpoint. The invention provides herewith a pharmaceutical composition comprising a nucleic acid, vector, host cell, or proteinaceous substance. In addition, such a pharmaceutical composition according to the invention is provided further comprising a nucleic acid encoding Apoptin or a functional equivalent or fragment thereof

[0027] Such a pharmaceutical composition is in particular provided for the induction of apoptosis, for example, wherein said apoptosis is p53-independent, for the treatment of a disease where enhanced cell proliferation or decreased cell death is observed, as is in general the case when said disease comprises cancer or autoimmune disease. Herewith the invention provides a method for treating an individual carrying a disease where enhanced cell proliferation or decreased cell death is observed comprising treating said individual with a pharmaceutical composition. In particular, these compositions comprise a factor of an apoptosis pathway, which is specific for transformed cells. Therefore, these compositions are essential for new treatments, but also for diagnosis of diseases related with aberrances in the apoptotic process, such as cancer, cancer-proneness and autoimmune diseases.

[0028] Furthermore, the invention provides a diagnostic assay based on the tumor-specific nuclear localization behavior of AAP-2, such as its dominant nuclear localization in human tumor cells but not in normal healthy cells.

[0029] The invention also provides an isolated or recombinant nucleic acid as set forth in SEQ ID NO.6 (FIG. 7). An isolated or recombinant proteinaceous substance encoded by a nucleic acid as set forth in SEQ ID NO.6 is also included.

[0030] The invention provides an isolated or recombinant proteinaceous substance comprising the amino acid sequence as set forth in SEQ ID NO.7 (FIG. 8).

[0031] The invention also provides an isolated or recombinant proteinaceous substance comprising the amino acid sequence as set forth in SEQ ID NO.8 (FIG. 9).

[0032] In a further embodiment, the invention provides an assay to identify a putative effector of the activity of the proteinaceous substance encoded by a nucleic acid as set forth in SEQ ID NO.6 (FIG. 7) comprising bringing in contact a proteinaceous substance comprising the amino acid sequence as set forth in SEQ ID NO.8 (FIG. 9) with said effector and determining the binding of said effector. Examples of methods to arrive at such an effector are binding studies, where an AAP-2 peptide comprising the PHD-finger domain (SEQ ID NO.8) (FIG. 9) is bound to a matrix and is tested whether test substances bind to the AAP-2 peptide, or by coimmunoprecipitation of an AAP-2 peptide comprising the PHD-finger domain with test substances using antibodies generated against the AAP-2 peptide comprising the PHD-finger domain. Test substances are, for example, small organic compounds derived, e.g., from a compound library or peptides or proteins derived, e.g., from a peptide library or from a natural source like a cell extract. The test substances are, for example, labeled for easier detection. The substances found to bind to the PHD-finger domain may either enhance or inhibit one or more effects of AAP-2. This is, for example, tested by measuring the apoptotic activity of AAP-2 as described above in the presence of said substances and by determining the nuclear localization of AAP-2 as described above in the presence of said substances. It is clear to a person skilled in the art that an assay to identify a putative effector of the activity of a proteinaceous substance encoded by a nucleic acid as set forth in SEQ ID NO.6 can also be performed with a functional equivalent or a functional fragment of SEQ ID NO.8 having the same kind of activity possibly in different amounts.

[0033] The invention will be explained in more detail in the following detailed description, which does not limit the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0034] We have used the yeast-2 hybrid system (Durfee et al., 1993) to identify Apoptin-associating cellular compounds, which are essential in the induction of apoptosis. The used system is an in vivo strategy to identify human proteins capable of physically associating with Apoptin. It has been used to screen cDNA libraries for clones encoding proteins capable of binding to a protein of interest (Fields and Song, 1989; Yang et al., 1992). The invention provides a, for example, novel Apoptin-associating protein, which is named Apoptin-associating protein 2 abbreviated as AAP-2. The invention also provides a method for inducing apoptosis through interference with the function of this newly discovered AAP-2 protein or functional equivalents or fragments thereof and/or the induction of apoptosis by means of (over)expression of AAP-2 or related gene or functional equivalents or fragments thereof. In addition, the invention also provides another Apoptin-associating protein, named AAP-3.

[0035] The invention also provides an antitumor therapy based on the interference with the function of AAP-2-like proteins and/or its (over)expression. An aberrant high level of AAP-2-like proteins will result in the induction of the opposite process of cell transformation, namely apoptosis. The invention furthermore provides a mediator of Apoptin-induced apoptosis, which is tumorspecific. The invention provides a therapy for cancer, autoimmune diseases or related diseases which is based on AAP-2-like proteins alone or in combination with Apoptin and/or Apoptin-like compounds.

Construction of pGBT9-VP3

[0036] For the construction of the bait plasmid, which enables the identification of Apoptin-associating proteins by means of a yeast-two-hybrid system, plasmid pET-1 6b-VP3 (Noteborn, unpublished results) was treated with NdeI and BamHI. The 0.4 kb NdeI-BamRI DNA fragment was isolated from low-melting-point agarose.

[0037] Plasmid pGBT9 (Clontech Laboratories, Inc., Palo Alto, USA) was treated with the restriction enzymes EcoRI and BamHI. The about 5.4-kb DNA fragment was isolated and ligated to an EcoRI-NdeI linker and the 0.4-kb DNA fragment containing the Apoptin-encoding sequences starting from its own ATG-initiation codon. The final construct containing a fusion gene of the GAL4-binding domain sequence and Apoptin under the regulation of the yeast promoter ADH was called pGBT-VP3 and was proven to be correct by restriction-enzyme analysis and DNA-sequencing according to the Sanger method (1977).

[0038] All cloning steps were essentially carried out as described by Maniatis et al. (1992). The plasmid pGBT-VP3 was purified by centrifugation in a CsCl gradient and column chromatography in Sephacryl S500 (Pharmacia).

GAL4-activation Domain-tagged cDNA Library

[0039] The expression vector pACT, containing the cDNAs from Epstein-Barr-virus-transformed human B cells fused to the GAL4 transcriptional activation domain, was used for detecting Apoptin-associating proteins. The pACT c-DNA library is derived from the lambda-ACT cDNA library, as described by Durfee et al. 1993.

Bacterial and Yeast Strains

[0040] The E. coli strain JM109 was the transformation recipient for the plasmid pGBT9 and pGBT-VP3. The bacterial strain electromax/DH10B was used for the transformation needed for the recovery of the Apoptin-associating pACT-cDNAs and was obtained from GIBCO-BRL, USA.

[0041] The yeast strain Y190 was used for screening the cDNA library and all other transformations, which are part of the used yeast-two-hybrid system

Media

[0042] For drug selections, Luria Broth (LB) plates for E. coli were supplemented with ampicillin (50 microgram per ml). Yeast YPD and SC media were prepared as described by Rose et al. (1990). Transformation of competent yeast strain Y190 with plasmids pGBT-VP3 and pACT-cDNA and screening for beta-galactosidase activity

[0043] The yeast strain Y190 was made competent and transformed according to the methods described by Klebe et al. (1983). The yeast cells were first transformed with pGBT-VP3 and subsequently transformed with pACT-cDNA, and these transformed yeast cells were grown on histidine-minus plates, also lacking leucine and tryptophan.

[0044] Hybond-N filters were layed on yeast colonies, which were histidine-positive and allowed to wet completely. The filters were lifted and submerged in liquid nitrogen to permeabilize the yeast cells. The filters were thawed and layed with the colony side up on Whattman 3MM paper in a petridish with Z-buffer (Per liter: 16.1 gr Na₂HPO₄.7H₂O, 5.5 gr NaH₂PO₄.H₂O, 0.75 gr KCl and 0,246 gr MgSO₄.7H₂O, pH 7.0) containing 0.27mercapto-ethanol and 1 mg/ml X-gal. The filters were incubated for at least 15 minutes or over night.

Recovery of Plasmids from Yeast

[0045] Total DNA from yeast cells, which were histidine- and beta-galactosidase-positive, was prepared by using the glusulase-alkaline lysis method as described by Hoffman and Winston (1987) and used to transform Electromax/DH10B bacteria via electroporation using a Bio-Rad GenePulser according the manufacturer's specifications.

[0046] Transformants were plated on LB media containing the antibiotic agent ampicilin.

Isolation of Apoptin-associating pACT Clones

[0047] By means of colony-filter assay, the colonies were lysed and hybridized to a radioactive-labeled 17-mer oligomer, which is specific for pACT (see also section Sequence analysis). Plasmid DNA was isolated from the pACT-clones and by means of Xhol digestion analysed for the presence of a cDNA insert.

Sequence Analysis

[0048] The subclone containing the sequence encoding the Apoptin-associating protein was partially sequenced using dideoxy NTPs according to the Sanger-method, which was performed by Eurogentec, Seraing, Belgium The used sequencing primer was a pACT-specific 17-mer comprising the DNA-sequence 5′-TACCACTACAATGGATG-3′ (SEQ ID NO.11).

[0049] The sequences of the Apoptin-associating cDNAs were compared with known gene sequences from the EMBL/Genbank.

Generation and Testing of Antibodies

[0050] In order to generate polyclonal antisera against the AAP-2 and AAP-3 protein, we designed three peptides per protein. These peptides were for AAP-2: 1) EVPKSTLESEKPGSP (SEQ. ID NO.12) (19-33) 2) ISSRKKKPDSPPKVL (SEQ. ID NO.13) (149-163) 3) TGSRTRGRWKYSSND (SEQ. ID NO.14) (256-270) The peptides for AAP-3 were: IYQRSGERPVTAGEE (SEQ. ID NO.15) (23-37) DEQVPDSIDAREIFD (SEQ. ID NO.16) (38-52) RSINDPEHPLTLEEL (SEQ. ID NO.17) (55-69)

[0051] The numbers in parenthesis correspond respectively to the amino acid sequences of FIG. 3 (SEQ. ID NO.3)and FIG. 6 (SEQ. ID NO.5).

[0052] These peptides were synthesized at Eurogentec (Belgium) with the standard addition of a C-terminal or N-terminal cysteine residue and all subsequent antibody syntheses were also performed there. These peptides were coupled to Keyhole Limpet Hemocyanin (KLH) and injected as a cocktail into two separate specific pathogen free rabbits with an immunization schedule of one injection and three subsequent boosts. Blood samples were taken before and after immunization. The sera were tested in-house for specific reactivity to the peptide cocktail by ELISA. The titers from each rabbit were high (>200,000). Furthermore, for certain subsequent purposes, the AAP-2 and AAP-3 antibodies were immune-purified using peptide cocktail coupled to immobilized diaminodipropylamine agarose columns (Pierce) according to the manufacturer's protocol.

[0053] The best AAP-2 and AAP-3 antibody preparation of the two generated was selected for further use. We tested the efficacy of this antibody by transfecting 6 cm plates of subconfluent primate COS-7 and human U₂OS cells using the calcium phosphate coprecipitation method with 5 μg of the AAP-2-myc or AAP-3-myc plasmid DNA construct and, as a control, untransfected cells. Two days post-transfection, cells were washed briefly in PBS, lysed in RIPA buffer (10 mM Tris 7.5, 150 mM NaCl, 0.1% SDS, 1.0% NP-49 and 1.0% sodium deoxycholate), clarified by centrifugation, and the supernatant fractionated on SDS-denaturing polyacrylamide gel electrophoresis. Proteins were Western-transferred to PVDF membranes (Immobilon, Millipore) using standard methodology. Membranes were blocked in 5% nonfat dry milk in tris-buffered saline containing 0.1% Tween-20, then incubated in the unpurified AAP-2 or AAP-3 antisera at a concentration of 1:5000. After a brief wash, membranes were further incubated in HRP-conjugated goat-anti-rabbit Ig at a concentration of 1:2000. After a thorough series of wash steps, proteins were detected using enhanced chemiluminescence (Amersham) according to the manufacturer's protocol and exposed to x-ray film and developed using standard automated machinery.

[0054] In addition, we tested the purified AAP-2 and AAP-3 antibody using immunoprecipitation in a manner the same as above, except that after centrifugation, the supernatant was added to 10 ul of AAP-2 or AAP-3 antibody precoupled to protein-A-sepharose beads, incubated for 1 hour with tumbling, then washed before fractionation on SDS-PAGE gels and Western analysis. Detection in this case was performed with the anti-myc tag monoclonal antibody 9E10 (Evan et al. 1985).

[0055] Finally, the purified antibody was tested for utility in immunofluorescence by including glass coverslips in the above transfections. Coverslips were fixed with 4% paraformaldehyde, blocked with normal goat serum, incubated in AAP-2 or AAP-3 antibody diluted 1:5, washed, incubated in FITC-conjugated goat-anti-rabbit Ig, mounted and visualized under fluorescence microscopy.

Results and Discussion

[0056] Apoptin induces specifically apoptosis in transformed cells, such as cell lines derived from human tumors. To identify the essential compounds in this cell-transformation-specific and/or tumor-specific apoptosis pathway, a yeast genetic screen was carried out.

[0057] We have used a human cDNA library, which is based on the plasmid vector pACT containing the complete cDNA copies made from Epstein-Barr virus-transformed human B cells (Durfee et al., 1993).

Construction of a Bait Plasmid Expressing a Fusion Gene Product of GAL4-DNA-binding Domain and Apoptin

[0058] To examine the existence of Apoptin-associating proteins in the human transformed/tumorigenic cDNA library, a so-called bait plasmid had to be constructed. To that end, the complete Apoptin-encoding region, flanked by about 40 basepairs downstream from the Apoptin gene, was cloned in the multiple cloning site of plasmid pGBT9.

[0059] The final construct, called pGBT-VP3, was analyzed by restriction-enzyme analysis and sequencing of the fusion area between Apoptin and the GAL4-DNA-binding domain.

A Gene(fragment) Encoding an Apoptin-associating Protein is Determined by Transactivation of a GAL4-responsive Promoter in Yeast

[0060] The Apoptin gene is fused to the GAL4-DNA-binding domain of plasmid pGBT-VP3, whereas all cDNAs derived from the transformed human B cells are fused to the GAL4-activation domain of plasmid pACT. If one of the proteinaceous substances encoded by said cDNAs binds to Apoptin, the GAL4-DNA-binding domain will be in the vicinity of the GAL4-activation domain resulting in the activation of the GAL4-responsive promoter, which regulates the reporter genes HIS3 and LacZ.

[0061] The yeast clones containing plasmid expressing Apoptin and a plasmid expressing an Apoptin-associating protein fragment can grow on a histidine-minus medium and will stain blue in a beta-galactosidase assay. Subsequently, the plasmid with the cDNA insert encoding the Apoptin-associating protein can be isolated and characterized.

[0062] Before we could do so, however, we have determined that transformation of yeast cells with pGBT-VP3 plasmid alone, or in combination with an empty pACT vector, did not result in the activation of the GALA-responsive promoter.

Identification of Apoptin-associating Protein Encoded by cDNA Derived from a Human Transformed B Cell Line

[0063] We have found two independent yeast colonies, which upon transformation with pGBT-VP3 and pACT-cDNA were able to grow on a histidine-minus medium (also lacking leucine and tryptophan) and stained blue in a beta-galactosidase assay. These results indicate that the observed yeast colonies contain, besides the bait plasmid pGBT-VP3, a pACT plasmid encoding a potential Apoptin-associating protein.

[0064] Plasmid DNA was isolated from the positive yeast colony, which was transformed in bacteria. By means of a filter-hybridization assay using a pACT-specific labeled DNA-probe, 2 independent clones containing pACT plasmid could be determined. Subsequently, pACT DNA was isolated and digested with restriction enzyme Xhol, which resulted in the presence of a 1.1-kbp (clone I) and a 1.3-kbp (clone II) cDNA insert, respectively. Finally, the pACT plasmids containing the two independent cDNA inserts were partially sequenced by using the Sanger method (Sanger et al., 1977).

Description of Apoptin-associating Proteins

[0065] The yeast genetic screen for Apoptin-associating proteins resulted in the detection of two cDNA clones comprising a single type of protein, namely a novel protein called Apoptin-associating protein 2, abbreviated as AAP-2.

[0066] The determined DNA sequence part of the AAP-2 cDNA clones AAP-2-I and AAP-2-II are shown in FIG. 1 (SEQ. ID NO.1) and 2 (SEQ. ID NO.2), respectively. The amino acid sequence, derived from the detected DNA sequence of clone AAP-2-II is given in FIG. 3 (SEQ. ID NO.3). Below the experiments will be described for AAP-2-II, which will be referred as AAP-2.

Construction of an Expression Vector for the Identification of AAP-2 Protein in Mammalian Cells

[0067] To study whether the cloned cDNA AAP-2 indeed encode (Apoptin-associating) a protein product, we have carried out the following experiments.

[0068] The DNA plasmid pMT2SM contains the adenovirus 5 major late promoter (MLP) and the SV40 ori enabling high levels of expression of foreign genes in transformed mammalian cells, such as SV-40-transformed Cos cells.

[0069] Furthermore, the pMT2SM vector contains a Myc-tag (amino acids: EQKLISEEDL) (SEQ. ID NO.18) which is in frame with the foreign-gene product. This Myc-tag enables the recognition of, e.g., Apoptin-associating proteins by means of the Myc-tag-specific 9E10 antibody.

[0070] The pMT2SM vector expressing Myc-tagged AAP-2 cDNA was constructed as follows. The pACT-AAP-2 cDNA clone was digested with the restriction enzyme Xhol and the cDNA insert was isolated. The expression vector pMT2SM was digested with XhoI and treated with calf intestine alkaline phosphatase and ligated to the isolated AAP-2 cDNA inserts. By sequence analysis, the pMT2SM constructs containing the AAP-2 cDNA in the correct orientation was identified.

[0071] The synthesis of Myc-tagged AAP-2 protein was analyzed by transfection of Cos cells with plasmid pMT2SM-AAP-2. As negative control, Cos cells were mock-transfected. Two days after transfection, the cells were lysed and Western-blot analysis was carried out using the Myc-tag-specific antibody 9E10.

[0072] The Cos cells transfected with pMT2SM-AAP-2 were proven to synthesize a specific Myc-tagged AAP-2 product with the size of approximately 70 kDa As expected, the lysates of the mock-transfected Cos cells did not contain a protein product reacting with the Myc-tag-specific antibodies.

[0073] These results indicate that we have been able to isolate a cDNA that is able to produce a protein product with the ability to associate to the apoptosis-inducing protein Apoptin.

Coimmunoprecipitation of Myc-tagged AAP-2 Protein with Apoptin in a Transformed Mammalian Cell System

[0074] Next, we have analyzed the association of Apoptin and the AAP-2 protein by means of coimmunoprecipitations using the Myc-tag-specific antibody 9E10. The 9E10 antibodies were shown not to bind directly to Apoptin, which enables the use of 9E10 for carrying out coimmunoprecipitations with (myc-tagged) Apoptin-associating proteins and Apoptin.

[0075] To that end, Cos cells were cotransfected with plasmid pCMV-VP3 encoding Apoptin and with plasmid pMT2SM-AAP-2. As a negative control, cells were transfected with pCMV-VP3 expressing Apoptin and a plasmid pcDNA3.1.LacZ-myc/His-LacZ encoding the myc-tagged betagalactosidase, which does not associate with Apoptin.

[0076] Two days after transfection, the cells were lysed in a buffer consisting of 50 mM Tris (7.5), 250 mM NaCl, 5 mM EDTA, 0.1% Triton X100, 1 mg/ml Na₄P₂O₇ and freshly added protease inhibitors such as PMSF, Tlypsine-inhibitor, Leupeptine and Na₃VO₄. The specific proteins were immunoprecipitated as described by Noteborn et al. (1998) using the Myc-tag-specific antibodies 9E10 and analyzed by Western blotting.

[0077] Staining of the Western blot with 9E10 antibodies and 111.3 antibodies, which are specifically directed against myc-tag and Apoptin, respectively, showed that the “total” cell lysates contained the 16-kDa Apoptin product and the Myc-tagged AAP-2 protein. By means of a specific LacZ polyclonal antibody, the beta-galactosidase product could be visualized.

[0078] Immunoprecipitation of the Myc-tagged AAP-2 products was accompanied by the immunoprecipatation of Apoptin product of 16 kDa. In contrast, immunoprecipitation of myc-tagged betagalactosidase did not result in a detectable coprecipitation of the Apoptin protein. In addition, immunoprecipitation of the Apoptin protein, by means of a polyclonal antibody directed against the C-terminal part of Apoptin (Noteborn and Danen, unpublished results) was accompanied by the immunoprecipitation of the AAP-2 product of approximately 70-kDa, but not by beta-galactosidase protein.

[0079] In total, three independent immunoprecipitation experiments were carried out, which all showed the specific associating ability of Apoptin protein to the AAP-2 protein.

[0080] These results indicate that the novel determined AAP-2 protein is able to specifically associate with Apoptin not only in the yeast background, but also in a mammalian transformed cellular system

Over-expression of the Novel AAP-2 Protein in Human Transformed Cells Induces the Apoptotic Process

[0081] In addition, we have examined whether AAP-2 carries apoptotic activity. First, we have analyzed the cellular localization of the novel AAP-2 protein in human transformed cells. To that end, the human osteosarcoma-derived Saos-2 cells were transfected, as described by Danen-van Oorschot (1997), with plasmid pMT2SM-AAP-2 encoding the myc-tagged AAP-2 protein, respectively.

[0082] By indirect immunofluorescence using the myc-tag-specific antibody 9E10 and DAPI, which stains the nuclear DNA, it was shown that AAP-2 protein was mainly present in the nucleus of most of the tumor cells and in a minor part of the cells both in the nucleus and cytoplasm or cytoplasm alone. These features suggest that, at least in human tumor cells, AAP-2 is involved in nuclear transport processes.

[0083] Already, three days after transfection, a significant amount of Saos-2 cells synthesizing AAP-2 underwent induction of apoptosis. These AAP-2-positive cells were aberrantly stained with DAPI, which is indicative for induction of apoptosis (Telford, 1992, Danen-van Oorschot, 1997). Cells expressing Apoptin also underwent apoptosis, whereas as expected the cells synthesizing the nonapoptotic betagalactosidase (LacZ) protein did not. Coexpression of Apoptin and AAP-2 protein in human tumor cells, such as Saos-2 cells, results in a slightly faster apoptotic process than as with the expression of Apoptin or AAP-2 protein alone. The results are shown in FIG. 4.

[0084] The fact that AAP-2 protein can induce apoptosis in p53-minus Saos-2 cells indicates that AAP-2 can induce p53-independent apoptosis. These results imply that AAP-2 can be used as an antitumor agent in cases where other (chemo)therapeutic agents will fail. Furthermore, the finding that both Apoptin and AAP-2 induce a p53-independent pathway indicates that AAP-2 fits in the Apoptin-induced apoptotic pathway.

[0085] In conclusion, we have identified an Apoptin-associating protein, namely the novel AAP-2 protein, which is mainly present in the nucleus and able to induce (p53-independent) apoptosis in human tumor cells.

AAP-2 does not Induce Apoptosis in Human Normal Diploid Cells

[0086] Next, we have examined whether AAP-2 behaves similar in normal human diploid nontransformed cells as has been found for AAP-2 in human tumor cells.

[0087] To that end, human diploid VH10 fibroblasts (Danen-Van Oorschot, 1997) were transfected using Fugene according the protocol of the supplier (Roche, Almere, The Netherlands) with plasmid pMT2SM-AAP-2b encoding the myc-tagged AAP protein. In parallel, human tumor-derived Saos-2 cells were also transfected with plasmid pMT2SM-AAP-2.

[0088] Three days after transfection, the cells were harvested and analyzed by indirect immunofluorescence using the myc-tag-specific antibody 9E10. Within the majority of AAP-2-positive human diploid cells, AAP-2 is located in the cytoplasm only or both in the nucleus and cytoplasm As expected, in most of the human tumor Saos-2 cells, AAP-2 is only located in the nucleus. Furthermore, the AAP-2-positive human normal diploid fibroblasts did not show a sign of AAP-2-induced apoptosis, as was examined by DAPI staining (see above).

[0089] In conclusion, we have identified an Apoptin-associating protein, namely AAP-2, which has a tumor-specific preference for induction of apoptosis and nuclear accumulation.

Further Sequence Analysis

[0090] A further sequence analysis of the human AAP-2 nucleic acid sequence yielded the 5331bp long nucleic acid sequence given in FIG. 7 (SEQ. ID NO.6). An open reading frame was found in this nucleic acid sequence at position 300-4499. The deduced amino acid sequence is given in FIG. 8 (SEQ. ID NO.7).

[0091] A protein domain called PHD-finger was found in the amino acid sequence of the human AAP-2 protein. It spans the region of amino acid 852 to amino acid 900. The Cys₄-His-Cys₃ zinc-finger-like motif which is characteristic for a PHD-finger domain (R. Aasland et al., 1995; TIBS 20, 56-59) is found in said region (see, FIG. 9).

[0092] The PHD-finger is found in nuclear proteins thought to be involved in chromatin-mediated transcriptional regulation. The PHD-finger was originally identified in a set of proteins that includes members of the Drosophila Polycomb and trithorax group genes. These genes regulate the expression of the homeotic genes through a mechanism thought to involve some aspect of chromatin structure. Other proteins which have this motif also have additional domains or characteristics that support that suggestion that the PHD-finger is involved in chromatin-mediated gene regulation. PHD-fingers are thought to be protein-protein interaction domains. Such protein-protein interactions are important for, e.g., the activity of multicomponent complexes involved in transcriptional activation or repression. PHD-fingers may also recognize a family of related targets in the nucleus such as the nucleosomal histone tails (R. Aasland et al., 1995; TIBS 20, 56-59).

[0093] PHD-finger domains are also found in a number of proteins closely associated with human tumorigenisis such as HRX/ALL1/MLL/Htrx, CBP, MOZ, all of which are part of aberrant fusion proteins derived from chromosomal translocations found in a high percentage of human leukemias (for review see Jacobson and Pillus, 1999; Current Opinion in Genetics & Dev. 9, 175-184). Other PHD-finger domain-containing proteins are overexpressed in certain tumor types (Lu, P. J. et al, 1999; J.Biol Chem 274, 15633-45).

[0094] Therefore, interfering with the functional activity of the PHD-finger domain of AAP-2 should have therapeutic effects against human tumors. The PHD-finger domain can be used to identify substances which bind to the PHD-finger domain. This can be done by methods known to persons skilled in the art, e.g., by binding studies, where an AAP-2 peptide comprising the PHD-finger domain is bound to a matrix and it is tested whether test substances bind to the AAP-2 peptide or by coimmunoprecipitation of an AAP-2 peptide comprising the PHD-finger domain with test substances using antibodies generated against the AAP-2 peptide comprising the PHD-finger domain. Test substances may be small organic compounds derived, e.g., from a compound library or peptides or proteins derived, e.g., from a peptide library or from a natural source like a cell extract. The test substances may be labeled for easier detection. The substances found to bind to the PHD-finger domain may either enhance or inhibit one or more effects of AAP-2. This can be tested by measuring the apoptotic activity of AAP-2 as described above in the presence of said substances and by determining the nuclear localization of AAP-2 as described above in the presence of said substances.

Another Apoptin-associating Protein

[0095] The genetic yeast screen with pGBT-VP3 as bait plasmid and pACT plasmid containing cDNAs from transformed human B cells also delivered the novel gene Apoptin-associating protein 3 (AAP-3). The DNA sequence of the AAP-3 is shown in FIG. 5, whereas the AAP-3 cDNA-encoded amino-acid sequence is shown in FIG. 6.

[0096] To analyze into further detail the associating properties of Apoptin and this AAP-3 protein, we have expressed a Myc-tagged AAP-3 cDNA by means of the pSM2NT vector (as described for AAP-2) in transformed mammalian Cos cells. Western blot analysis using the Myc-tag-specific antibodies 9E10 showed a specific (Myc-tagged) AAP-3 protein of approximately 22-kDa. This major 22-kDa AAP-3 product is accompanied by smaller and larger minor AAP-3-specific products. These results indicate that the isolated cDNA indeed encodes a protein of the expected size.

[0097] Next, immunoprecipitation assays were carried out with transiently transfected Cos cells cosynthesizing Myc-tagged AAP-3 and Apoptin. The results clearly showed that both 9E10 antibodies and Apoptin-specific 111.3 antibodies precipitate AAP-3 protein and Apoptin, which indicates that Apoptin associates with this new AAP-3 protein in a mammalian transformed background. In total, three independent immunoprecipitation experiments were carried out, which all showed the associating ability of Apoptin to the AAP-3 protein.

[0098] Immunofluorescence assays of human transformed Saos-2 cells and normal diploid VH10 fibroblasts expressing AAP-3 revealed that AAP-3 is located in both cell types predominantly in the cytoplasm and nucleus, but in lower percentages also mainly in the nucleus or mainly in the cytoplasm Cosynthesis of AAP-3 and Apoptin in both cell types showed a clear perinuclear colocalization of AAP-3 and Apoptin. Tumor cells that have become apoptotic showed a nuclear localization of Apoptin and a perinuclear stainings pattern of AAP-3. As expected, normal diploid VH10 cells synthesizing both Apoptin and AAP-3 did not undergo apoptosis.

[0099] These data indicate that AAP-3 will release Apoptin when the cell has become tumorigenic and/or transformed, resulting in the nuclear localization of Apoptin and induction of apoptosis.

[0100] In summary, our findings prove that our newly discovered AAP-3 protein is able to associate to the tumor-specific apoptosis-inducing protein Apoptin in both a yeast and mammalian cellular background. Therefore, this AAP-3 protein plays an important role in the induction of (Apoptin-regulated) tumors-specific apoptosis.

Utility of AAP-2 and AAP-3 Antisera

[0101] The best AAP-2 and AAP-3 antibody preparations obtained from the two rabbit derived antisera were selected for further use. We tested the efficacy of these antibody preparations against AAP-2 and AAP-3, respectively, by transfecting primate COS-7 and human U₂OS cells with the AAP-2-myc or AAP-3-myc construct. Western analysis showed that the approximately 70 kDa AAP-2-myc protein and the approximately 22 kDa AAP-3-myc were detected strongly only in samples where the DNA was transfected. Similarly, in immunoprecipitation experiments, AAP-2-myc or AAP-3-myc protein was also strongly detected. Finally, localization of AAP-2-nyc or AAP-3-myc protein in a cell using the AAP-2 or AAP-3 antibody could be determined by immunofluorescence analysis.

Overexpression of 2 Apoptin-associating Proteins

[0102] The genetic yeast screen with pGBT-VP3 as bait plasmid and pACT plasmid containing cDNAs from transformed human B cells also delivered another gene, which also encodes an Apoptin-associating protein. This Apoptin-associating protein was called AAP-4 (see copending application EP00204396.6, which is incorporated herein by reference). The nucleic acid sequence of AAP-4 is shown in FIG. 10 (SEQ. ID NO.9). An open reading frame was found in this nucleic acid sequence at position 236 to 2866. The deduced amino acid sequence is given in FIG. 11 (SEQ. ID NO.10). Just like AAP-2 and AAP-3, AAP-4 is able to associate with Apoptin not only in the yeast background, but also in a mammalian transformed cellular system Furthermore, this protein is present in the nucleus and able to induce (p53-independent) apoptosis in human tumor cells. A functional equivalent or a functional fragment of AAP-4 is herein also included. A functional equivalent or a functional fragment of AAP-4 is a derivative or a fragment having the same kind of activity possibly in different amounts. It is clear to a person skilled in the art that there are different ways of arriving at a functional equivalent or functional fragment. A functional equivalent can, for example, be a point mutant or a deletion mutant or a equivalent derived from another species. Another way to arrive at a functional equivalent is a molecular evolution of equivalents and/or fragments having the same kind of activity possibly in different amounts.

[0103] To study whether two separate Apoptin-associating proteins can not only bind to Apoptin but also to another Apoptin-associating protein, we carried out the following experiment.

[0104] Immunoprecipitation assays were carried out with transiently transfected Cos cells cosynthesizing Myc-tagged AAP-3 and Myc-tagged AAP-4. The results clearly showed that antibodies directed against AAP-3 and antibodies directed against AAP-4 both precipitate AAP-3 and AAP-4, which suggests that AAP-3 and AAP-4 associate in this mammalian transformed background. In total, three independent immunoprecipitation experiments were carried out, which all showed the associating ability of AAP-3 and AAP-4.

[0105] The fact that two proteins, which showed to be Apoptin-associating proteins can independently coassociate in the absence of Apoptin strengthens the idea that the AAP-3/Apoptin coassociation is physiologically relevant.

Diagnostic Assay for Cancer Cells

[0106] Based on the present report, we can conclude that the cellular localization of AAP-2 is different in tumorigenic/transformed human cells in comparison to normal human nontransformed cells. Furthermore, accumulation of AAP-2 in the nucleus correlates with apoptosis induction, whereas cytoplasmic/nuclear localization correlates with cell viability and normal proliferative capacity. Therefore, we are able to develop a diagnostic assay for the identification of (human) cancer cells versus normal “healthy” nontransformed cells.

[0107] The assay consists of transfecting “suspicious” (human) cells, for instance from human origin, with a plasmid encoding AAP-2 or infecting the cells with viral vectors expressing AAP-2. Subsequently, the cells will be examined 1) for the ability to undergo apoptosis by the over-expressing AAP-2 gene and 2) for a main shift in the localization of AAP-2 from the cytoplasm to the nucleus.

[0108] The intracellular localization of AAP-2 can be determined, using an immunofluorescence assay with monoclonal antibodies specific for AAP-2 and/or specific for a tag linked to AAP-2 such as the herein described myc-tag. If the percentage of apoptosis and/or the nuclear localization of AAP-2 in the analyzed cells expressing AAP-2 is significantly higher than in AAP-2-positive control “healthy” cells, one can conclude that the analyzed cells has become tumorigenic/transformed. As positive control known human tumorigenic cells will be used for expressing AAP-2.

Coexpression of SV40 large T Antigen and AAP-2 results in Translocation of AAP-2 and Induction of Apoptosis

[0109] We have examined the effect of expression of transforming genes on AAP-2-induced apoptosis in normal human cells derived from healthy individuals. To that end, human VH10 diploid fibroblasts were transiently cotransfected with plasmid pMT2SM-AAP-2 encoding AAP-2 protein and either plasmid pR-s884 encoding SV40 large T antigen, or the negative-control plasmid pCMV-neo (Noteborn and Zhang, 1998).

[0110] By indirect immunofluorescence, the cells were analyzed for AAP-2-induced apoptosis. The normal VH10 cells did not undergo apoptosis when AAP-2 was transfected with the negative-control plasmid. The results showed, as expected, that expression of AAP-2 is not able to induce apoptosis in normal human diploid cells, confirming the above mentioned data. However, normal diploid human fibroblasts expressing both AAP-2 and SV40 large T antigen underwent AAP-2-induced apoptosis.

[0111] The transition of normal human cells, from AAP-2-resistance to AAP-2-susceptibility, can probably be explained by the fact that the AAP-2 protein translocates from a cytoplasmic localization to a nuclear localization. This transition becomes apparent already 2 days after transfection of plasmids encoding the transforming protein SV40 large T antigen. One can conclude that an event takes place, in this example due to expression of a transforming product derived from a DNA-tumor virus, which results in the translocation of over-expressed AAP-2 from the cytoplasm to the nucleus, which is followed by induction of apoptosis.

Diagnostic Assay for Cancer-inducing Genes, Agents and Cancer-proneness Based on AAP-2-Induced Apoptosis

[0112] Based on the present report, we are able to develop a diagnostic assay for the identification of cancer-inducing and/or transforming agents or genes.

[0113] A first type of assay consists of transfecting “normal” cells, for instance from human origin, with a plasmid encoding AAP-2, or infecting the cells with viral vectors expressing AAP-2, together with a plasmid encoding a putative transforming/cancer-inducing gene. Subsequently, the cells will be examined 1) for the ability to undergo apoptosis by the over-expressing AAP-2 gene and 2) for a shift in the localization of AAP-2 from the cytoplasm to the nucleus.

[0114] The intracellular localization of AAP-2 can be determined using an immunofluorescence assay with monoclonal antibodies specific for AAP-2 and/or specific for a tag linked to AAP-2 such as the herein described myc-tag. If the percentage of apoptosis and/or the nuclear localization of AAP-2 in normal cells coexpressing AAP-2 and the putative transforming/cancer-inducing gene is significantly higher than in AAP-2-positive control cells expressing a control plasmid, one can conclude that the analyzed gene indeed has transforming/cancer-inducing activity.

[0115] A second example of a diagnostic test is based on the treatment of cultured normal diploid cells with a putative carcinogenic agent. The agent can be added, for instance, to the culture medium for various lengths of time. Subsequently, the cells are transfected with a plasmid encoding AAP-2. This approach can also be carried out by first transfecting/infecting the normal diploid cells and then treating the cells with the agent to be tested. The subsequent steps of the assay are the same as the first type of diagnostic assay described in this section. If the percentage of apoptosis and/or the nuclear localization of AAP-2 in normal cells expressing AAP-2 and the putative carcinogenic agent is significantly higher than in AAP-2-positive control cells expressing a control agent, one can conclude that the analyzed agent indeed has transforming/cancer-inducing activity.

[0116] A third example of a diagnostic test is based on the treatment of cultured normal diploid cells derived from a skin biopsy of the potential cancer-prone individual to be tested and cultured in suitable medium. Next, the cells are irradiated with UV and subsequently transfected with a plasmid encoding AAP-2 or infected with a viral vector expressing AAP-2 or the cells are first transfected and/or infected and then irradiated. In parallel, diploid cells from a normal healthy individual will be used as a control.

[0117] The subsequent steps of the assay are the same as the first type of diagnostic assay described in this section. If after V-treatment the percentage of apoptosis and/or the nuclear localization of AAP-2 in diploid cells derived from the potential cancer-prone individual is significantly higher than in UV-treated AAP-2-positive control cells, one can conclude that the analyzed cells are cancer-proneness cells.

DESCRIPTION OF THE FIGURES

[0118]FIG. 1 shows the partial sequence of vector pMT2SM-AAP-2-I.

[0119]FIG. 2 shows the partial sequence of vector pMT2SM-AAP-2-II.

[0120]FIG. 3 shows the amino-acid sequence of the analyzed region of the Apoptin-associating clone AAP-2-II. In addition, the three C-terminal amino acids H-E-G (bold) of the multiple cloning site of pACT are given to illustrate that the AAP-2 amino acid sequence is in frame with the GAL4-activation domain. This feature proves that the AAP-2-II region is indeed synthesized in yeast cells.

[0121]FIG. 4 shows the apoptotic activity of AAP-2 protein and/or Apoptin in human osteosarcoma-derived Saos-2 cells. (−): no apoptotic activity; (+): apoptotic activity; (++): strong apoptotic activity; (+++): very strong apoptotic activity. In total three independent experiments have been carried out.

[0122]FIG. 5 shows the partial sequence of vector pMT2SM-AAP-3.

[0123]FIG. 6 shows the amino-acid sequence of the analyzed region of the Apoptin-associating clone AAP-3. In addition, the three C-terminal amino acids H-E-G (bold) of the multiple cloning site of pACT are given to illustrate that the AAP-3 amino acid sequence is in frame with the GAL4-activation domain. This feature proves that the AAP-3 region is indeed synthesized in yeast cells.

[0124]FIG. 7 shows the nucleic acid sequence of AAP-2.

[0125]FIG. 8 shows the amino acid sequence deduced from the nucleic acid sequence of FIG. 7.

[0126]FIG. 9 shows the PHD-finger domain of the AAP-2 protein

[0127]FIG. 10 shows the nucleic acid sequence of AAP-4.

[0128]FIG. 11 shows the amino acid sequence deduced from the nucleic acid sequence of FIG. 10.

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1 18 1 873 DNA Homo sapiens misc_feature Description of Sequence partial sequence of vector pMT2SM-AAP-2- 1 cccagacctc tggcatagag gagccttctg agacaaaggg ttctatgcaa aaaagcaaat 60 tcaaatataa gttggttcct gaagaagaaa ccactgcctc agaaaataca gagataacct 120 ctgaaaggca gaaagagggc atcaaattaa caatcaggat atcaagtcgg aaaaagaagc 180 ccgattctcc ccccaaagtt ctagaaccag aaaacaagca agagaagaca gaaaaggaag 240 aggagaaaac aaatgtgggt cgtactttaa gaagatctcc aagaatatct agacccactg 300 caaaggtggc tgagatcaga gatcagaaag ctgataaaaa aagaggggaa ggagaagatg 360 aggtggaaga agagtcaaca gctttgcaaa aaactgacaa aaaggaaatt ttgaaaaaat 420 cagagaaaga tacaaattct aaagtaagca aggtaaaacc caaaggcaaa gttcgatgga 480 ctggttctcg gacacgtggc agatggaaat attccagcaa tgatgaaagt gaagggtctg 540 gcagtgaaaa atcatctgca gcttcagaag aggaggagga aaaggaaagt gaagaagcca 600 tcctagcaga tgatgatgaa ccatgcaaaa aatgtggcct tccaaaccat cctgagctaa 660 ttcttctgtg tgactcttgc gatagtggat accatactgc ctgccttcgc cctcctctga 720 tgatcatccc agatggagaa tggttctgcc caccttgcca acataaactg ctctgtgaaa 780 aattagagga acagttgcag gatttggatg ttgccttaaa gaagaaagag cgtgccgaac 840 gaagaaaaga acgcttggtg tatgttggta tca 873 2 1150 DNA Homo sapiens misc_feature Description of sequence partial sequence of vector pMT2SM-AAP-2-I 2 ccttttcttg ataaggacgc acaaagattg agtccaatac cagaagaagt tccaaagagt 60 actctagagt cagaaaagcc tggctctcct gaggcagctg aaacttctcc accatctaat 120 atcattgacc actgtgagaa actagcctca gaaaaagaag tggtagaatg ccagagtaca 180 agtactgttg gtggccagtc tgtgaaaaaa gtagacctag aaaccctaaa agaggattct 240 gagttcacaa aggtagaaat ggataatctg gacaatgccc agacctctgg catagaggag 300 ccttctgaga caaagggttc tatgcaaaaa agcaaattca aatataagtt ggttcctgaa 360 gaagaaacca ctgcctcaga aaatacagag ataacctctg aaaggcagaa agagggcatc 420 aaattaacaa tcaggatatc aagtcggaaa aagaagcccg attctccccc caaagttcta 480 gaaccagaaa acaagcaaga gaagacagaa aaggaagagg agaaaacaaa tgtgggtcgt 540 actttaagaa gatctccaag aatatctaga cccactgcaa aggtggctga gatcagagat 600 cagaaagctg ataaaaaaag aggggaagga gaagatgagg tggaagaaga gtcaacagct 660 ttgcaaaaaa ctgacaaaaa ggaaattttg aaaaaatcag agaaagatac aaattctaaa 720 gtaagcaagg taaaacccaa aggcaaagtt cgatggactg gttctcggac acgtggcaga 780 tggaaatatt ccagcaatga tgaaagtgaa gggtctggca gtgaaaaatc atctgcagct 840 tcagaagagg aggaggaaaa ggaaagtgaa gaagccatcc tagcagatga tgatgaacca 900 tgcaaaaaat gtggccttcc aaaccatcct gagctaattc ttctgtgtga ctcttgcgat 960 agtggatacc atactgcctg ccttcgccct cctctgatga tcatcccaga tggagaatgg 1020 ttctgcccac cttgccaaca taaactgctc tgtgaaaaat tagaggaaca gttgcaggat 1080 ttggatgttg ccttaaagaa gaaagagcgt gccgaacgaa gaaaagaacg cttggtgtat 1140 gttggtatca 1150 3 386 PRT Homo sapiens misc_feature Description of Sequence amino acid sequence of the analyzed region of the Apoptin-associating clone AAP-2-II 3 His Glu Gly Pro Phe Leu Asp Lys Asp Ala Gln Arg Leu Ser Pro Ile 1 5 10 15 Pro Glu Glu Val Pro Lys Ser Thr Leu Glu Ser Glu Lys Pro Gly Ser 20 25 30 Pro Glu Ala Ala Glu Thr Ser Pro Pro Ser Asn Ile Ile Asp His Cys 35 40 45 Glu Lys Leu Ala Ser Glu Lys Glu Val Val Glu Cys Gln Ser Thr Ser 50 55 60 Thr Val Gly Gly Gln Ser Val Lys Lys Val Asp Leu Glu Thr Leu Lys 65 70 75 80 Glu Asp Ser Glu Phe Thr Lys Val Glu Met Asp Asn Leu Asp Asn Ala 85 90 95 Gln Thr Ser Gly Ile Glu Glu Pro Ser Glu Thr Lys Gly Ser Met Gln 100 105 110 Lys Ser Lys Phe Lys Tyr Lys Leu Val Pro Glu Glu Glu Thr Thr Ala 115 120 125 Ser Glu Asn Thr Glu Ile Thr Ser Glu Arg Gln Lys Glu Gly Ile Lys 130 135 140 Leu Thr Ile Arg Ile Ser Ser Arg Lys Lys Lys Pro Asp Ser Pro Pro 145 150 155 160 Lys Val Leu Glu Pro Glu Asn Lys Gln Glu Lys Thr Glu Lys Glu Glu 165 170 175 Glu Lys Thr Asn Val Gly Arg Thr Leu Arg Arg Ser Pro Arg Ile Ser 180 185 190 Arg Pro Thr Ala Lys Val Ala Glu Ile Arg Asp Gln Lys Ala Asp Lys 195 200 205 Lys Arg Gly Glu Gly Glu Asp Glu Val Glu Glu Glu Ser Thr Ala Leu 210 215 220 Gln Lys Thr Asp Lys Lys Glu Ile Leu Lys Lys Ser Glu Lys Asp Thr 225 230 235 240 Asn Ser Lys Val Ser Lys Val Lys Pro Lys Gly Lys Val Arg Trp Thr 245 250 255 Gly Ser Arg Thr Arg Gly Arg Trp Lys Tyr Ser Ser Asn Asp Glu Ser 260 265 270 Glu Gly Ser Gly Ser Glu Lys Ser Ser Ala Ala Ser Glu Glu Glu Glu 275 280 285 Glu Lys Glu Ser Glu Glu Ala Ile Leu Ala Asp Asp Asp Glu Pro Cys 290 295 300 Lys Lys Cys Gly Leu Pro Asn His Pro Glu Leu Ile Leu Leu Cys Asp 305 310 315 320 Ser Cys Asp Ser Gly Tyr His Thr Ala Cys Leu Arg Pro Pro Leu Met 325 330 335 Ile Ile Pro Asp Gly Glu Trp Phe Cys Pro Pro Cys Gln His Lys Leu 340 345 350 Leu Cys Glu Lys Leu Glu Glu Gln Leu Gln Asp Leu Asp Val Ala Leu 355 360 365 Lys Lys Lys Glu Arg Ala Glu Arg Arg Lys Glu Arg Leu Val Tyr Val 370 375 380 Gly Ile 385 4 651 DNA Homo sapiens misc_feature Description of Sequence partial sequence of vector pMT2SM-AAP-3 4 ccgatggtag gcggcggcgg ggtcggcggc ggcctcctgg agaatgccaa ccccctcatc 60 taccagcgct ctggggagcg gcctgtgacg gcaggcgagg aggacgagca ggttcccgac 120 agcatcgacg cacgcgagat cttcgarctg attcgctcca tcaatgaccc ggagcatcca 180 ctgacgctag aggagttgaa cgtagtagag caggtgcggg ttcaggttag cgaccccgag 240 agtacagtgg ctgtggcttt cacaccaacc attccgcact gcagcatggc cacccttatt 300 ggtctgtcca tcaaggtcaa gcttctgcgc tcccttcctc agcgtttcaa gatggacgtg 360 cacattactc cggggaccca tgcctcagag catgcagtga acaagcaact tgcagataag 420 gagcgggtgg cagctgccct ggagaacacc cacctcttgg aggttgtgaa tcagtgcctg 480 tcagcccgct cctgagcctg gcctttgacc cctcaacctg catactgggt atcctggtcc 540 caactcctgc caagggctgt taccgttgtt ttcctggaat cactcacaaa tgagaaacta 600 acatttgcct ttttgtaata aagttaattt atattcaaaa aaaaaaaaaa c 651 5 167 PRT Homo sapiens misc_feature Description of Sequence amino acid sequence of the analyzed region of the Apoptin-associating clone AAP- 5 His Glu Gly Pro Met Val Gly Gly Gly Gly Val Gly Gly Gly Leu Leu 1 5 10 15 Glu Asn Ala Asn Pro Leu Ile Tyr Gln Arg Ser Gly Glu Arg Pro Val 20 25 30 Thr Ala Gly Glu Glu Asp Glu Gln Val Pro Asp Ser Ile Asp Ala Arg 35 40 45 Glu Ile Phe Asp Leu Ile Arg Ser Ile Asn Asp Pro Glu His Pro Leu 50 55 60 Thr Leu Glu Glu Leu Asn Val Val Glu Gln Val Arg Val Gln Val Ser 65 70 75 80 Asp Pro Glu Ser Thr Val Ala Val Ala Phe Thr Pro Thr Ile Pro His 85 90 95 Cys Ser Met Ala Thr Leu Ile Gly Leu Ser Ile Lys Val Lys Leu Leu 100 105 110 Arg Ser Leu Pro Gln Arg Phe Lys Met Asp Val His Ile Thr Pro Gly 115 120 125 Thr His Ala Ser Glu His Ala Val Asn Lys Gln Leu Ala Asp Lys Glu 130 135 140 Arg Val Ala Ala Ala Leu Glu Asn Thr His Leu Leu Glu Val Val Asn 145 150 155 160 Gln Cys Leu Ser Ala Arg Ser 165 6 5331 DNA Homo sapiens misc_feature Description of Sequence nucleic acid sequence of AAP-2 6 ggcaagcccg aagggaagga gagaaggggg cggaaagagg gcggaaagtg aaaggcgccg 60 agggccgctc tgtctcccgt ctgactcggt tctcgactgc tccgggccgc cgatgtattg 120 tgggatcgcg gaccgtccct gagacgctgg gatccgcaga ggagcccact tgagagcgcc 180 tcctgtcgtc tgtaaggttg ccttgccatc cctcggcacc ccaacttccc ccgccccccc 240 atcgcctcct cctccatcct ccagttcagg cggcgcaggg cggcggcacg gcggcggtga 300 tggctcctcc gggctgcccg ggttcgtgcc ccaacttcgc cgtagtctgc tccttcttgg 360 agcgctacgg gccgctgcta gacctgcctg agttgccgtt ccctgagctg gagcgggtgc 420 tgcaggcgcc gccgccggac gtcggcaacg gagaagtacc aaaagaattg gtggagctcc 480 atttgaagct gatgaggaaa attggcaaat ctgttactgc agacagatgg gaaaaatatt 540 tgatcaagta cctctgtgag tgtcagtttg atgacaatct caaattcaag aatattatta 600 atgaggagga tgccgatact atgcgtctcc agccaattgg tcgagacaaa gatggcctca 660 tgtactggta ccaattggat caagatcaca atgtcagaat gtacatagaa gaacaagatg 720 atcaagatgg ctcttcatgg aaatgcattg tcagaaatcg aaacgagttg gctgagactc 780 ttgcactcct gaaagcacaa attgatcctg tactattgaa aaactctagc caacaagaca 840 actcttctcg ggaaagtccc agcttagagg atgaggagac taaaaaagag gaagaaacac 900 ctaaacaaga ggaacagaaa gaaagtgaaa agatgaaaag tgaggagcag cctatggatt 960 tagaaaaccg ttctacagcc aatgttctag aagagactac tgtgaaaaaa gaaaaagaag 1020 atgaaaagga acttgtgaaa ctgccagtca tagtgaagct agaaaaacct ttgccagaaa 1080 atgaagaaaa aaagattatc aaagaagaaa gtgattcctt caaggaaaat gtcaaaccca 1140 ttaaagttga ggtgaaggaa tgtagagcag atcctaaaga taccaaaagt agcatggaga 1200 agccagtggc acaggagcct gaaaggatcg aatttggtgg caatattaaa tcttctcacg 1260 aaattactga gaaatctact gaagaaactg agaaacttaa aaatgaccag caggccaaga 1320 taccactaaa aaaacgagaa attaaactga gtgatgattt tgacagtcca gtcaagggac 1380 ctttgtgtaa atcagttact ccaacaaaag agtttttgaa agatgaaata aaacaagagg 1440 aagagacttg taaaaggatc tctacaatca ctgctttggg tcatgaaggg aaacagctgg 1500 taaatggaga agttagtgat gaaagggtag ctccaaattt taagacagaa ccaatagaga 1560 caaagtttta tgagacaaag gaagagagct atagcccctc taaggacaga aatatcatca 1620 cggagggaaa tggaacagag tccttaaatt ctgtcataac aagtatgaaa acaggtgagc 1680 ttgagaaaga aacagcccct ttgaggaaag atgcagatag ttcaatatca gtcttagaga 1740 tccatagtca aaaagcacaa atagaggaac ccgatcctcc agaaatggaa acttctcttg 1800 attcttctga gatggcaaaa gatctctctt caaaaactgc tttatcttcc accgagtcgt 1860 gtaccatgaa aggtgaagag aagtctccca aaactaagaa ggataagcgc ccaccaatcc 1920 tagaatgtct tgaaaagtta gagaagtcca aaaagacttt tcttgataag gacgcacaaa 1980 gattgagtcc aataccagaa gaagttccaa agagtactct agagtcagaa aagcctggct 2040 ctcctgaggc agctgaaact tctccaccat ctaatatcat tgaccactgt gagaaactag 2100 cctcagaaaa agaagtggta gaatgccaga gtacaagtac tgttggtggc cagtctgtga 2160 aaaaagtaga cctagaaacc ctaaaagagg attctgagtt cacaaaggta gaaatggata 2220 atctggacaa tgcccagacc tctggcatag aggagccttc tgagacaaag ggttctatgc 2280 aaaaaagcaa attcaaatat aagttggttc ctgaagaaga aaccactgcc tcagaaaata 2340 cagagataac ctctgaaagg cagaaagagg gcatcaaatt aacaatcagg atatcaagtc 2400 ggaaaaagaa gcccgattct ccccccaaag ttctagaacc agaaaacaag caagagaaga 2460 cagaaaagga agaggagaaa acaaatgtgg gtcgtacttt aagaagatct ccaagaatat 2520 ctagacccac tgcaaaagtg gctgagatca gagatcagaa agctgataaa aaaagagggg 2580 aaggagaaga tgaggtggaa gaagagtcaa cagctttgca aaaaactgac aaaaaggaaa 2640 ttttgaaaaa atcagagaaa gatacaaatt ctaaagtaag caaggtaaaa cccaaaggca 2700 aagttcgatg gactggttct cggacacgtg gcagatggaa atattccagc aatgatgaaa 2760 gtgaagggtc tggcagtgaa aaatcatctg cagcttcaga agaggaggaa gaaaaggaaa 2820 gtgaagaagc catcctagca gatgatgatg aaccatgcaa aaaatgtggc cttccaaacc 2880 atcctgagct aattcttctg tgtgactctt gcgatagtgg ataccatact gcctgccttc 2940 gccctcctct gatgatcatc ccagatggag aatggttctg cccaccttgc caacataaac 3000 tgctctgtga aaaattagag gaacagttgc aggatttgga tgttgcctta aagaagaaag 3060 agcgtgccga acgaagaaaa gaacgcttgg tgtatgttgg tatcagtatt gaaaacatca 3120 ttcctccaca agagccagac ttttctgaag atcaagaaga aaagaaaaaa gattcaaaaa 3180 aatccaaagc aaacttgctt gaaaggaggt caacaagaac aaggaaatgt ataagctaca 3240 gatttgatga gtttgatgaa gcaattgatg aagctattga agatgacatc aaagaagccg 3300 atggaggagg agttggccga ggaaaagata tctccaccat cacaggtcat cgtgggaaag 3360 acatctctac tattttggat gaagaaagaa aagaaaataa acgaccccag agggcagctg 3420 ctgctcgaag gaagaaacgc cggcgattaa atgatctgga cagtgatagc aacctggatg 3480 aagaagagag cgaggatgaa ttcaagatca gtgatggatc tcaagatgag tttgttgtgt 3540 ctgatgaaaa cccagatgaa agtgaagaag atccgccatc taatgatgac agtgacactg 3600 acttttgtag ccgtagactg aggcgacacc cctctcggcc aatgaggcag agcaggcgtt 3660 tgcgaagaaa gaccccaaag aaaaaatatt ccgatgatga tgaagaggag gaatctgagg 3720 agaatagtag agactctgaa agtgacttca gtgatgattt tagtgatgat tttgtagaaa 3780 ctcggcgaag gcggtcaagg agaaatcaga aaagacaaat taactacaaa gaagactcag 3840 aaagtgacgg nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3900 gaagactttc cagctcagag agtgaagaga gctatttgtc caagaactct gaagatgatg 3960 agctagctaa agaatcaaag cggtcagttc gaaagcgggg ccgaagcaca gacgagtatt 4020 cagaagcaga tgaggaggag gaggaagagg aaggcaaacc atcccgcaaa cggctacacc 4080 ggattgagac ggatgaggag gagagttgtg acaatgctca tggagatgca aatcagcctg 4140 cccgtgacag ccagcctagg gtcctgccct cagaacaaga gagcaccaag aagccctacc 4200 ggatagaaag tgatgaggaa gaggactttg aaaatgtagg caaagtgggg agcccattgg 4260 actatagctt agtggactta ccttcaacca atggacagag ccctggcaaa gccattgaga 4320 acttgattgg caagcctact gagaagtctc agacccccaa ggacaacagc acagccagtg 4380 caagcctagc ctccaatggg acaagtggtg ggcaggaggc aggagcacca gaagaggagg 4440 aagatgagct tttgagagtg actgaccttg ttgattatgt ctgtaacagt gaacagttat 4500 aagacttttt ttccattttt gtgctaattt attccacggt agctctcaca ccagcgggcc 4560 agttattaaa agctgtttaa tttttcctag aaaactccac tacagaatga cttttagaag 4620 aaaaatttca acaaatcctg aagtctttct gtgaagtgac cagttctgaa ctttgaagat 4680 aaataattgc tgtaaattcc ttttgatttt ctttttccag gttcatggtc cttggtaatt 4740 tcattcatgg aaaaaaatct tattataata acaacaaaga tttgtatatt tttgacttta 4800 tatttcctga gctctcctga ctttgtgaaa aagggtggat gaaaatgcat tccgaatctg 4860 tgagggccca aaacagaatt taggggtggg tgaaagcact tgtgctttag ctttttcata 4920 ttaaatatat attatattta aacattcatg gcatagatga tgatttacag acaatttaaa 4980 agttcaagtc tgtactgtta cagtttgaga attgtagata acatcataca taagtcattt 5040 agtaacagcc tttgtgaaat gaacttgttt actattggag ataaccacac ttaataaaga 5100 agagacagtg aaagtaccat cataattaac ctaaattttt gttatagcag agtttcttgt 5160 ttaaaaaaaa ataaaatcat ctgaaaagca aaaatacagt aaaatgtata atgaagcttt 5220 gccaaccaga ctgtgctagc aacaaatttt tttaaataag ctttatgcag tggtaataag 5280 gtggcctcaa atatattgtg tctgatggag agttattagt gaaatgaatg t 5331 7 1400 PRT Homo sapiens misc_feature Description of Sequence Amino acid sequence deduced from the nucleic acid seqeuence of AAP- 7 Met Ala Pro Pro Gly Cys Pro Gly Ser Cys Pro Asn Phe Ala Val Val 1 5 10 15 Cys Ser Phe Leu Glu Arg Tyr Gly Pro Leu Leu Asp Leu Pro Glu Leu 20 25 30 Pro Phe Pro Glu Leu Glu Arg Val Leu Gln Ala Pro Pro Pro Asp Val 35 40 45 Gly Asn Gly Glu Val Pro Lys Glu Leu Val Glu Leu His Leu Lys Leu 50 55 60 Met Arg Lys Ile Gly Lys Ser Val Thr Ala Asp Arg Trp Glu Lys Tyr 65 70 75 80 Leu Ile Lys Tyr Leu Cys Glu Cys Gln Phe Asp Asp Asn Leu Lys Phe 85 90 95 Lys Asn Ile Ile Asn Glu Glu Asp Ala Asp Thr Met Arg Leu Gln Pro 100 105 110 Ile Gly Arg Asp Lys Asp Gly Leu Met Tyr Trp Tyr Gln Leu Asp Gln 115 120 125 Asp His Asn Val Arg Met Tyr Ile Glu Glu Gln Asp Asp Gln Asp Gly 130 135 140 Ser Ser Trp Lys Cys Ile Val Arg Asn Arg Asn Glu Leu Ala Glu Thr 145 150 155 160 Leu Ala Leu Leu Lys Ala Gln Ile Asp Pro Val Leu Leu Lys Asn Ser 165 170 175 Ser Gln Gln Asp Asn Ser Ser Arg Glu Ser Pro Ser Leu Glu Asp Glu 180 185 190 Glu Thr Lys Lys Glu Glu Glu Thr Pro Lys Gln Glu Glu Gln Lys Glu 195 200 205 Ser Glu Lys Met Lys Ser Glu Glu Gln Pro Met Asp Leu Glu Asn Arg 210 215 220 Ser Thr Ala Asn Val Leu Glu Glu Thr Thr Val Lys Lys Glu Lys Glu 225 230 235 240 Asp Glu Lys Glu Leu Val Lys Leu Pro Val Ile Val Lys Leu Glu Lys 245 250 255 Pro Leu Pro Glu Asn Glu Glu Lys Lys Ile Ile Lys Glu Glu Ser Asp 260 265 270 Ser Phe Lys Glu Asn Val Lys Pro Ile Lys Val Glu Val Lys Glu Cys 275 280 285 Arg Ala Asp Pro Lys Asp Thr Lys Ser Ser Met Glu Lys Pro Val Ala 290 295 300 Gln Glu Pro Glu Arg Ile Glu Phe Gly Gly Asn Ile Lys Ser Ser His 305 310 315 320 Glu Ile Thr Glu Lys Ser Thr Glu Glu Thr Glu Lys Leu Lys Asn Asp 325 330 335 Gln Gln Ala Lys Ile Pro Leu Lys Lys Arg Glu Ile Lys Leu Ser Asp 340 345 350 Asp Phe Asp Ser Pro Val Lys Gly Pro Leu Cys Lys Ser Val Thr Pro 355 360 365 Thr Lys Glu Phe Leu Lys Asp Glu Ile Lys Gln Glu Glu Glu Thr Cys 370 375 380 Lys Arg Ile Ser Thr Ile Thr Ala Leu Gly His Glu Gly Lys Gln Leu 385 390 395 400 Val Asn Gly Glu Val Ser Asp Glu Arg Val Ala Pro Asn Phe Lys Thr 405 410 415 Glu Pro Ile Glu Thr Lys Phe Tyr Glu Thr Lys Glu Glu Ser Tyr Ser 420 425 430 Pro Ser Lys Asp Arg Asn Ile Ile Thr Glu Gly Asn Gly Thr Glu Ser 435 440 445 Leu Asn Ser Val Ile Thr Ser Met Lys Thr Gly Glu Leu Glu Lys Glu 450 455 460 Thr Ala Pro Leu Arg Lys Asp Ala Asp Ser Ser Ile Ser Val Leu Glu 465 470 475 480 Ile His Ser Gln Lys Ala Gln Ile Glu Glu Pro Asp Pro Pro Glu Met 485 490 495 Glu Thr Ser Leu Asp Ser Ser Glu Met Ala Lys Asp Leu Ser Ser Lys 500 505 510 Thr Ala Leu Ser Ser Thr Glu Ser Cys Thr Met Lys Gly Glu Glu Lys 515 520 525 Ser Pro Lys Thr Lys Lys Asp Lys Arg Pro Pro Ile Leu Glu Cys Leu 530 535 540 Glu Lys Leu Glu Lys Ser Lys Lys Thr Phe Leu Asp Lys Asp Ala Gln 545 550 555 560 Arg Leu Ser Pro Ile Pro Glu Glu Val Pro Lys Ser Thr Leu Glu Ser 565 570 575 Glu Lys Pro Gly Ser Pro Glu Ala Ala Glu Thr Ser Pro Pro Ser Asn 580 585 590 Ile Ile Asp His Cys Glu Lys Leu Ala Ser Glu Lys Glu Val Val Glu 595 600 605 Cys Gln Ser Thr Ser Thr Val Gly Gly Gln Ser Val Lys Lys Val Asp 610 615 620 Leu Glu Thr Leu Lys Glu Asp Ser Glu Phe Thr Lys Val Glu Met Asp 625 630 635 640 Asn Leu Asp Asn Ala Gln Thr Ser Gly Ile Glu Glu Pro Ser Glu Thr 645 650 655 Lys Gly Ser Met Gln Lys Ser Lys Phe Lys Tyr Lys Leu Val Pro Glu 660 665 670 Glu Glu Thr Thr Ala Ser Glu Asn Thr Glu Ile Thr Ser Glu Arg Gln 675 680 685 Lys Glu Gly Ile Lys Leu Thr Ile Arg Ile Ser Ser Arg Lys Lys Lys 690 695 700 Pro Asp Ser Pro Pro Lys Val Leu Glu Pro Glu Asn Lys Gln Glu Lys 705 710 715 720 Thr Glu Lys Glu Glu Glu Lys Thr Asn Val Gly Arg Thr Leu Arg Arg 725 730 735 Ser Pro Arg Ile Ser Arg Pro Thr Ala Lys Val Ala Glu Ile Arg Asp 740 745 750 Gln Lys Ala Asp Lys Lys Arg Gly Glu Gly Glu Asp Glu Val Glu Glu 755 760 765 Glu Ser Thr Ala Leu Gln Lys Thr Asp Lys Lys Glu Ile Leu Lys Lys 770 775 780 Ser Glu Lys Asp Thr Asn Ser Lys Val Ser Lys Val Lys Pro Lys Gly 785 790 795 800 Lys Val Arg Trp Thr Gly Ser Arg Thr Arg Gly Arg Trp Lys Tyr Ser 805 810 815 Ser Asn Asp Glu Ser Glu Gly Ser Gly Ser Glu Lys Ser Ser Ala Ala 820 825 830 Ser Glu Glu Glu Glu Glu Lys Glu Ser Glu Glu Ala Ile Leu Ala Asp 835 840 845 Asp Asp Glu Pro Cys Lys Lys Cys Gly Leu Pro Asn His Pro Glu Leu 850 855 860 Ile Leu Leu Cys Asp Ser Cys Asp Ser Gly Tyr His Thr Ala Cys Leu 865 870 875 880 Arg Pro Pro Leu Met Ile Ile Pro Asp Gly Glu Trp Phe Cys Pro Pro 885 890 895 Cys Gln His Lys Leu Leu Cys Glu Lys Leu Glu Glu Gln Leu Gln Asp 900 905 910 Leu Asp Val Ala Leu Lys Lys Lys Glu Arg Ala Glu Arg Arg Lys Glu 915 920 925 Arg Leu Val Tyr Val Gly Ile Ser Ile Glu Asn Ile Ile Pro Pro Gln 930 935 940 Glu Pro Asp Phe Ser Glu Asp Gln Glu Glu Lys Lys Lys Asp Ser Lys 945 950 955 960 Lys Ser Lys Ala Asn Leu Leu Glu Arg Arg Ser Thr Arg Thr Arg Lys 965 970 975 Cys Ile Ser Tyr Arg Phe Asp Glu Phe Asp Glu Ala Ile Asp Glu Ala 980 985 990 Ile Glu Asp Asp Ile Lys Glu Ala Asp Gly Gly Gly Val Gly Arg Gly 995 1000 1005 Lys Asp Ile Ser Thr Ile Thr Gly His Arg Gly Lys Asp Ile Ser 1010 1015 1020 Thr Ile Leu Asp Glu Glu Arg Lys Glu Asn Lys Arg Pro Gln Arg 1025 1030 1035 Ala Ala Ala Ala Arg Arg Lys Lys Arg Arg Arg Leu Asn Asp Leu 1040 1045 1050 Asp Ser Asp Ser Asn Leu Asp Glu Glu Glu Ser Glu Asp Glu Phe 1055 1060 1065 Lys Ile Ser Asp Gly Ser Gln Asp Glu Phe Val Val Ser Asp Glu 1070 1075 1080 Asn Pro Asp Glu Ser Glu Glu Asp Pro Pro Ser Asn Asp Asp Ser 1085 1090 1095 Asp Thr Asp Phe Cys Ser Arg Arg Leu Arg Arg His Pro Ser Arg 1100 1105 1110 Pro Met Arg Gln Ser Arg Arg Leu Arg Arg Lys Thr Pro Lys Lys 1115 1120 1125 Lys Tyr Ser Asp Asp Asp Glu Glu Glu Glu Ser Glu Glu Asn Ser 1130 1135 1140 Arg Asp Ser Glu Ser Asp Phe Ser Asp Asp Phe Ser Asp Asp Phe 1145 1150 1155 Val Glu Thr Arg Arg Arg Arg Ser Arg Arg Asn Gln Lys Arg Gln 1160 1165 1170 Ile Asn Tyr Lys Glu Asp Ser Glu Ser Asp Gly Ser Gln Lys Ser 1175 1180 1185 Leu Arg Arg Gly Lys Glu Ile Arg Arg Val His Lys Arg Arg Leu 1190 1195 1200 Ser Ser Ser Glu Ser Glu Glu Ser Tyr Leu Ser Lys Asn Ser Glu 1205 1210 1215 Asp Asp Glu Leu Ala Lys Glu Ser Lys Arg Ser Val Arg Lys Arg 1220 1225 1230 Gly Arg Ser Thr Asp Glu Tyr Ser Glu Ala Asp Glu Glu Glu Glu 1235 1240 1245 Glu Glu Glu Gly Lys Pro Ser Arg Lys Arg Leu His Arg Ile Glu 1250 1255 1260 Thr Asp Glu Glu Glu Ser Cys Asp Asn Ala His Gly Asp Ala Asn 1265 1270 1275 Gln Pro Ala Arg Asp Ser Gln Pro Arg Val Leu Pro Ser Glu Gln 1280 1285 1290 Glu Ser Thr Lys Lys Pro Tyr Arg Ile Glu Ser Asp Glu Glu Glu 1295 1300 1305 Asp Phe Glu Asn Val Gly Lys Val Gly Ser Pro Leu Asp Tyr Ser 1310 1315 1320 Leu Val Asp Leu Pro Ser Thr Asn Gly Gln Ser Pro Gly Lys Ala 1325 1330 1335 Ile Glu Asn Leu Ile Gly Lys Pro Thr Glu Lys Ser Gln Thr Pro 1340 1345 1350 Lys Asp Asn Ser Thr Ala Ser Ala Ser Leu Ala Ser Asn Gly Thr 1355 1360 1365 Ser Gly Gly Gln Glu Ala Gly Ala Pro Glu Glu Glu Glu Asp Glu 1370 1375 1380 Leu Leu Arg Val Thr Asp Leu Val Asp Tyr Val Cys Asn Ser Glu 1385 1390 1395 Gln Leu 1400 8 46 PRT Homo sapiens misc_feature Description of Sequence PHD-finger domain of the AAP-2 proetin 8 Glu Pro Cys Lys Lys Cys Gly Leu Pro Asn His Pro Glu Leu Ile Leu 1 5 10 15 Leu Cys Asp Ser Cys Asp Ser Gly Tyr His Thr Ala Cys Leu Arg Pro 20 25 30 Pro Leu Met Ile Ile Pro Asp Gly Glu Trp Phe Cys Pro Pro 35 40 45 9 5690 DNA Homo sapiens misc_feature Description of Sequence nucleic acid sequence of AAP-4 9 cggcagggca gcggggcgat gaggtgagga cgcccgggaa ccggaggcgg caccgcgcgg 60 cgcacggacc tgggacgcgg agtcctgaag ccggcggacg gttttcgtac gggcggccgt 120 gcgcgaggcg aggagagaac attgaaagta ttctctaagc tatttgaaga gagtgactaa 180 atgcacctgg gtcaggctgt ctgtgggtat gaagtggttg ggagaatcca agaacatggt 240 ggtgaatggc aggagaaatg gaggcaagtt gtctaatgac catcagcaga atcaatcaaa 300 attacagcac acggggaagg acaccctgaa ggctggcaaa aatgcagtcg agaggaggtc 360 gaacagatgt aatggtaact cgggatttga aggacagagt cgctatgtac catcctctgg 420 aatgtccgcc aaggaactct gtgaaaatga tgacctagca accagtttgg ttcttgatcc 480 ctatttaggt tttcaaacac acaaaatgaa tactagcgcc tttccttcga ggagctcaag 540 gcatttttca aaatctgaca gtttttctca caacaaccct gtgagattta ggcctattaa 600 aggaaggcag gaagaactaa aggaagtaat tgaacgtttt aagaaagatg aacacttgga 660 gaaagccttc aaatgtttga cttcaggcga atgggcacgg cactattttc tcaacaagaa 720 taaaatgcag gagaaattat tcaaagaaca tgtatttatt tatttgcgaa tgtttgcaac 780 tgacagtgga tttgaaatat tgccatgtaa tagatactca tcagaacaaa atggagccaa 840 aatagttgca acaaaagagt ggaaacgaaa tgacaaaata gaattactgg tgggttgtat 900 tgccgaactt tcagaaattg aggagaacat gctacttaga catggagaaa acgacttcag 960 tgtcatgtac tccacaagga aaaactgtgc tcaactctgg ctgggtcctg ctgcgtttat 1020 aaaccatgat tgcagaccta attgtaagtt tgtgtcaact ggtcgagata cagcatgtgt 1080 gaaggctcta agagacattg aacctggaga agaaatttct tgttattatg gagatgggtt 1140 ctttggagaa aataatgagt tctgcgagtg ttacacttgc gaaagacggg gcactggtgc 1200 ttttaaatcc agagtgggac tgcctgcgcc tgctcctgtt atcaatagca aatatggact 1260 cagagaaaca gataaacgtt taaataggct taaaaagtta ggtgacagca gcaaaaattc 1320 agacagtcaa tctgtcagct ctaacactga tgcagatacc actcaggaaa aaaacaatgc 1380 aacttctaac cgaaaatctt cagttggcgt aaaaaagaat agcaagagca gaacgttaac 1440 gaggcaatct atgtcaagaa ttccagcttc ttccaactct acctcatcta agctaactca 1500 tataaataat tccagggtac caaagaaact gaagaagcct gcaaagcctt tactttcaaa 1560 gataaaattg agaaatcatt gcaagcggct ggagcaaaag aatgcttcaa gaaaactcga 1620 aatgggaaac ttagtactga aagagcctaa agtagttctg tataaaaatt tgcccattaa 1680 aaaagataag gagccagagg gaccagccca agccgcagtt gccagcgggt gcttgactag 1740 acacgcggcg agagaacaca gacagaatcc tgtgagaggt gctcattcgc agggggagag 1800 ctcgccctgc acctacataa ctcggcggtc agtgaggaca agaacaaatc tgaaggaggc 1860 ctctgacatc aagcttgaac caaatacgtt gaatggctat aaaagcagtg tgacggaacc 1920 ttgccccgac agtggtgaac agctgcagcc agctcctgtg ctgcaggagg aagaactggc 1980 tcatgagact gcacaaaaag gggaggcaaa gtgtcataag agtgacacag gcatgtccaa 2040 aaagaagtca cgacaaggaa aacttgtgaa acagtttgca aaaatagagg aatctactcc 2100 agtgcacgat tctcctggaa aagacgacgc ggtaccagat ttgatgggtc cccattctga 2160 ccagggtgag cacagtggca ctgtgggcgt gcctgtgagc tacacagact gtgctccttc 2220 acccgtcggt tgttcagttg tgacatcaga tagcttcaaa acaaaagaca gctttagaac 2280 tgcaaaaagt aaaaagaaga ggcgaatcac aaggtatgat gcacagttaa tcctagaaaa 2340 taactctggg attcccaaat tgactcttcg taggcgtcat gatagcagca gcaaaacaaa 2400 tgaccaagag aatgatggaa tgaactcttc caaaataagc atcaagttaa gcaaagacca 2460 tgacaacgat aacaatctct atgtagcaaa gcttaataat ggatttaact caggatcagg 2520 cagtagttct acaaaattaa aaatccagct aaaacgagat gaggaaaata gggggtctta 2580 tacagagggg cttcatgaaa atggggtgtg ctgcagtgat cctctttctc tcttggagtc 2640 tcgaatggag gtggatgact atagtcagta tgaggaagaa agtacagatg attcctcctc 2700 ttctgagggc gatgaagagg aggatgacta tgatgatgac tttgaagacg attttattcc 2760 tcttcctcca gctaagcgct tgaggttaat agttggaaaa gactctatag atattgacat 2820 ttcttcaagg agaagagaag atcagtcttt aaggcttaat gcctaagctc ttggtcttaa 2880 cttgacctgg gataactact ttaaagaaat aaaaaattcc agtcaattat tcctcaactg 2940 aaagtttagt ggcagcactt ctattgtccc ttcacttatc agcatactat tgtagaaagt 3000 gtacagcata ctgactcaat tcttaagtct gatttgtgca aatttttatc gtacttttta 3060 aatagccttc ttacgtgcaa ttctgagtta gaggtaaagc cctgttgtaa aataaaggct 3120 caagcaaaat tgtacagtga tagcaacttt ccacacagga cgttgaaaac agtaatgtgg 3180 ctacacagtt tttttaactg taagagcatc agctggctct ttaatatatg actaaacaat 3240 aatttaaaac aaatcatagt agcagcatat taagggtttc tagtatgcta atatcaccag 3300 caatgatctt tggctttttg atttatttgc tagatgtttc ccccttggag ttttgtcagt 3360 ttcacactgt ttgctggccc aggtgtactg tttgtggcct ttgttaatat cgcaaaccat 3420 tggttgggag tcagattggt ttcttaaaaa aaaaaaaaaa atgacatacg tgacagctca 3480 cttttcagtt cattatatgt acgagggtag cagtgtgtgg gatgaggttc gatacagcgt 3540 atttattgct tgtcatgtaa attaaaaacc ttgtatttaa ctcttttcaa tccttttaga 3600 taaaattgtt ctttgcaaga atgattggtg cttatttttt caaaaatttg ctgtgaacaa 3660 cgtgatgaca acaagcaaca tttatctaat gaactacagc tatcttaatt tggttcttca 3720 agttttctgt tgcacttgta aaatgctaca aggaatatta aaaaaatcta ttcactttaa 3780 cttataatag tttatgaaat aaaaacatga gtcacagctt ttgttctgtg gtaacctata 3840 aaaaaagttt gtctttgaga ttcaatgtaa agaactgaaa acaatgtata tgttgtaaat 3900 atttgtgtgt tgtgagacat ttttgtcata agaaattaaa agaacttacc aggaaggttt 3960 ttaagtttag aaatattcat gccaataaaa taggaaatta taaatatata gttttaagca 4020 ctgcatcagt gggagttctt ggcttatgtt agtttatgtt agtttattat gaaaacatca 4080 aagatttttt tgactatatt atcagttaaa caaaaaggag tcagatttaa tttgtttttt 4140 gaagcacttt gagaaattaa ttttaattaa cttaatgagc aaatttttat tactacttta 4200 tgttcaatac caggttcttt tcatttctct ggattatttt gcaaatcatt ggacagagaa 4260 tttgggaata taaatctgta acaggtgttg acaccagtag gtctctttat ttctgggaaa 4320 tgtgtacctg tactttctga tatacagtgt tcctaagtaa aaatcaattc aggggatttg 4380 tatagtgtct ataggaaagt agcccatgtc ttgaaatatg aaaaggaatc tgaaggtcat 4440 gaaaagtcca gtggagaaaa tctcaatgct tactgttact actaattgat tcctactagt 4500 ttccaggttt ggggggatat tgtttcaatg acgctcctta agactgttga ttgcccatag 4560 gttccaaata gaaattaaga ctcatgaaca tttttagaaa gtagattgtt ttctcctggt 4620 tctctaagga actacttctg cagtcttaca tagtctcatc cttgtttgtt gtggtgcagt 4680 cgaactcctc aggcgtttgg aaagcatgtg gtagaccttc ttccacaccc acccataccc 4740 ccgttcactg cgtctggagg tcttcaacag tgaagtaggg cagcccacac agcctctcag 4800 gagcacctgt ccgaggcacc cggagcactt tgcagagcac gtccagccct catggggtcc 4860 ctgcatagaa atgtgaaccc ctgccactga ggaagatgaa ggtagaccct gtgtctggag 4920 gtgctggagg gcagcgggtc acctcttgta ttcccacctt agtttggggt gttttgaaga 4980 ggttcagaga ctaaatctta aaccttattt gaataccaac gatagctatt ttgggaattt 5040 cgatcttaaa aagtgacaaa acacatttcc cattttcatt tttcagctga attttagtaa 5100 cttatttttg atgttttaat tttatcatgg cctcctcttt ggaggccaac cttcccatgg 5160 gtctcaaagc agtgacattt ggtagtaaat cactgcctct caggagtcgg tatgcacaag 5220 cactcagcag ccactgttga tgccttctag ggaaacctaa tttccgttgg taaaggtagg 5280 ggcctcggaa ctgttccgga tctgctgtag aacttcaccg tgtggaatgg tgacagccac 5340 acaccgttga ccagtttaga agaggttgca ttcaataaaa ctcttagctt gagcttatgc 5400 aatgattggt taagattttg gcattgtaag aattaggaga tgatcataga aatatatgta 5460 aagtattcaa ttttcaatca ttttcaaatt actgttataa attgtttttg ctgagttgta 5520 atacttttga gatacaatgt attccttgta ctgaaagaat gaaaaaggac tttttcagca 5580 tttgaggtaa gttctttaac gtttcattaa aaacattttt tacaaatatt ttgtacatgc 5640 acttgcagta ttgaggttaa tcattttaat aaattcggaa attaaaaaaa 5690 10 876 PRT Homo sapiens misc_feature Description of Sequence Amino acid sequence deduced from the nucleic acid seqeuence of AAP- 10 Met Val Val Asn Gly Arg Arg Asn Gly Gly Lys Leu Ser Asn Asp His 1 5 10 15 Gln Gln Asn Gln Ser Lys Leu Gln His Thr Gly Lys Asp Thr Leu Lys 20 25 30 Ala Gly Lys Asn Ala Val Glu Arg Arg Ser Asn Arg Cys Asn Gly Asn 35 40 45 Ser Gly Phe Glu Gly Gln Ser Arg Tyr Val Pro Ser Ser Gly Met Ser 50 55 60 Ala Lys Glu Leu Cys Glu Asn Asp Asp Leu Ala Thr Ser Leu Val Leu 65 70 75 80 Asp Pro Tyr Leu Gly Phe Gln Thr His Lys Met Asn Thr Ser Ala Phe 85 90 95 Pro Ser Arg Ser Ser Arg His Phe Ser Lys Ser Asp Ser Phe Ser His 100 105 110 Asn Asn Pro Val Arg Phe Arg Pro Ile Lys Gly Arg Gln Glu Glu Leu 115 120 125 Lys Glu Val Ile Glu Arg Phe Lys Lys Asp Glu His Leu Glu Lys Ala 130 135 140 Phe Lys Cys Leu Thr Ser Gly Glu Trp Ala Arg His Tyr Phe Leu Asn 145 150 155 160 Lys Asn Lys Met Gln Glu Lys Leu Phe Lys Glu His Val Phe Ile Tyr 165 170 175 Leu Arg Met Phe Ala Thr Asp Ser Gly Phe Glu Ile Leu Pro Cys Asn 180 185 190 Arg Tyr Ser Ser Glu Gln Asn Gly Ala Lys Ile Val Ala Thr Lys Glu 195 200 205 Trp Lys Arg Asn Asp Lys Ile Glu Leu Leu Val Gly Cys Ile Ala Glu 210 215 220 Leu Ser Glu Ile Glu Glu Asn Met Leu Leu Arg His Gly Glu Asn Asp 225 230 235 240 Phe Ser Val Met Tyr Ser Thr Arg Lys Asn Cys Ala Gln Leu Trp Leu 245 250 255 Gly Pro Ala Ala Phe Ile Asn His Asp Cys Arg Pro Asn Cys Lys Phe 260 265 270 Val Ser Thr Gly Arg Asp Thr Ala Cys Val Lys Ala Leu Arg Asp Ile 275 280 285 Glu Pro Gly Glu Glu Ile Ser Cys Tyr Tyr Gly Asp Gly Phe Phe Gly 290 295 300 Glu Asn Asn Glu Phe Cys Glu Cys Tyr Thr Cys Glu Arg Arg Gly Thr 305 310 315 320 Gly Ala Phe Lys Ser Arg Val Gly Leu Pro Ala Pro Ala Pro Val Ile 325 330 335 Asn Ser Lys Tyr Gly Leu Arg Glu Thr Asp Lys Arg Leu Asn Arg Leu 340 345 350 Lys Lys Leu Gly Asp Ser Ser Lys Asn Ser Asp Ser Gln Ser Val Ser 355 360 365 Ser Asn Thr Asp Ala Asp Thr Thr Gln Glu Lys Asn Asn Ala Thr Ser 370 375 380 Asn Arg Lys Ser Ser Val Gly Val Lys Lys Asn Ser Lys Ser Arg Thr 385 390 395 400 Leu Thr Arg Gln Ser Met Ser Arg Ile Pro Ala Ser Ser Asn Ser Thr 405 410 415 Ser Ser Lys Leu Thr His Ile Asn Asn Ser Arg Val Pro Lys Lys Leu 420 425 430 Lys Lys Pro Ala Lys Pro Leu Leu Ser Lys Ile Lys Leu Arg Asn His 435 440 445 Cys Lys Arg Leu Glu Gln Lys Asn Ala Ser Arg Lys Leu Glu Met Gly 450 455 460 Asn Leu Val Leu Lys Glu Pro Lys Val Val Leu Tyr Lys Asn Leu Pro 465 470 475 480 Ile Lys Lys Asp Lys Glu Pro Glu Gly Pro Ala Gln Ala Ala Val Ala 485 490 495 Ser Gly Cys Leu Thr Arg His Ala Ala Arg Glu His Arg Gln Asn Pro 500 505 510 Val Arg Gly Ala His Ser Gln Gly Glu Ser Ser Pro Cys Thr Tyr Ile 515 520 525 Thr Arg Arg Ser Val Arg Thr Arg Thr Asn Leu Lys Glu Ala Ser Asp 530 535 540 Ile Lys Leu Glu Pro Asn Thr Leu Asn Gly Tyr Lys Ser Ser Val Thr 545 550 555 560 Glu Pro Cys Pro Asp Ser Gly Glu Gln Leu Gln Pro Ala Pro Val Leu 565 570 575 Gln Glu Glu Glu Leu Ala His Glu Thr Ala Gln Lys Gly Glu Ala Lys 580 585 590 Cys His Lys Ser Asp Thr Gly Met Ser Lys Lys Lys Ser Arg Gln Gly 595 600 605 Lys Leu Val Lys Gln Phe Ala Lys Ile Glu Glu Ser Thr Pro Val His 610 615 620 Asp Ser Pro Gly Lys Asp Asp Ala Val Pro Asp Leu Met Gly Pro His 625 630 635 640 Ser Asp Gln Gly Glu His Ser Gly Thr Val Gly Val Pro Val Ser Tyr 645 650 655 Thr Asp Cys Ala Pro Ser Pro Val Gly Cys Ser Val Val Thr Ser Asp 660 665 670 Ser Phe Lys Thr Lys Asp Ser Phe Arg Thr Ala Lys Ser Lys Lys Lys 675 680 685 Arg Arg Ile Thr Arg Tyr Asp Ala Gln Leu Ile Leu Glu Asn Asn Ser 690 695 700 Gly Ile Pro Lys Leu Thr Leu Arg Arg Arg His Asp Ser Ser Ser Lys 705 710 715 720 Thr Asn Asp Gln Glu Asn Asp Gly Met Asn Ser Ser Lys Ile Ser Ile 725 730 735 Lys Leu Ser Lys Asp His Asp Asn Asp Asn Asn Leu Tyr Val Ala Lys 740 745 750 Leu Asn Asn Gly Phe Asn Ser Gly Ser Gly Ser Ser Ser Thr Lys Leu 755 760 765 Lys Ile Gln Leu Lys Arg Asp Glu Glu Asn Arg Gly Ser Tyr Thr Glu 770 775 780 Gly Leu His Glu Asn Gly Val Cys Cys Ser Asp Pro Leu Ser Leu Leu 785 790 795 800 Glu Ser Arg Met Glu Val Asp Asp Tyr Ser Gln Tyr Glu Glu Glu Ser 805 810 815 Thr Asp Asp Ser Ser Ser Ser Glu Gly Asp Glu Glu Glu Asp Asp Tyr 820 825 830 Asp Asp Asp Phe Glu Asp Asp Phe Ile Pro Leu Pro Pro Ala Lys Arg 835 840 845 Leu Arg Leu Ile Val Gly Lys Asp Ser Ile Asp Ile Asp Ile Ser Ser 850 855 860 Arg Arg Arg Glu Asp Gln Ser Leu Arg Leu Asn Ala 865 870 875 11 17 DNA Artificial Sequence misc_feature Description of Artificial Sequence primer specific for pACT 11 taccactaca atggatg 17 12 15 PRT Artificial Sequence misc_feature Description of Artificial Sequence peptide based on sequence of AAP-2 protei 12 Glu Val Pro Lys Ser Thr Leu Glu Ser Glu Lys Pro Gly Ser Pro 1 5 10 15 13 15 PRT Artificial Sequence misc_feature Description of Artificial Sequence peptide based on sequence of AAP-2 protei 13 Ile Ser Ser Arg Lys Lys Lys Pro Asp Ser Pro Pro Lys Val Leu 1 5 10 15 14 15 PRT Artificial Sequence misc_feature Description of Artificial Sequence peptide based on sequence of AAP-2 protei 14 Thr Gly Ser Arg Thr Arg Gly Arg Trp Lys Tyr Ser Ser Asn Asp 1 5 10 15 15 15 PRT Artificial Sequence misc_feature Description of Artificial Sequence peptide based on sequence of AAP-3 protei 15 Ile Tyr Gln Arg Ser Gly Glu Arg Pro Val Thr Ala Gly Glu Glu 1 5 10 15 16 15 PRT Artificial Sequence misc_feature Description of Artificial Sequence peptide based on sequence of AAP-3 protei 16 Asp Glu Gln Val Pro Asp Ser Ile Asp Ala Arg Glu Ile Phe Asp 1 5 10 15 17 15 PRT Artificial Sequence misc_feature Description of Artificial Sequence peptide based on sequence of AAP-3 protei 17 Arg Ser Ile Asn Asp Pro Glu His Pro Leu Thr Leu Glu Glu Leu 1 5 10 15 18 10 PRT Artificial Sequence misc_feature Description of Artificial Sequence Myc-tag 18 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 

1. An isolated or recombinant nucleic acid or functional equivalent or functional fragment thereof encoding an Apoptin-associating proteinaceous substance capable of providing apoptosis.
 2. The isolated or recombinant nucleic acid of claim 1 derived from a cDNA library.
 3. (Twice Amended) The isolated or recombinant nucleic acid of claim 2 wherein said cDNA library comprises human cDNA.
 4. (Twice Amended) The isolated or recombinant nucleic acid of claim 1 capable of hybridizing to a nucleic acid molecule encoding the Apoptin-associating proteinaceous substance as shown in FIG. 1, 2, 5 or
 7. 5. (Twice Amended) The isolated or recombinant nucleic acid of claim 1 being at least 60% homologous to a nucleic acid molecule, or to a functional equivalent or functional fragment thereof, encoding the Apoptin-associating proteinaceous substance as shown in FIG. 1, 2, 5 or
 7. 6. (Previously Amended) A vector comprising the nucleic acid of claim
 5. 7. The vector of claim 6 further comprising a gene-delivery vehicle.
 8. (Previously Amended) A host cell comprising the nucleic acid of claim
 1. 9. The host cell of claim 8 wherein said cell is a eukaryotic cell.
 10. (Previously Amended) An isolated or recombinant Apoptin-associating proteinaceous substance capable of providing apoptosis encoded by the nucleic acid of claim
 1. 12. (Previously Amended) The proteinaceous substance of claim 10 comprising at least a part of an amino acid sequence as shown in FIG. 3, 6 or 8 or a functional equivalent or functional fragment thereof.
 13. (Twice Amended) An isolated or synthetic antibody specifically recognizing the proteinaceous substance or the functional equivalent or the functional fragment thereof according to claim
 12. 15. (Twice Amended) A method of inducing apoptosis in a cell, said method comprising: introducing the proteinaceous substance of claim 12 to the cell in a manner to induce said apoptosis.
 16. The method according to claim 15 wherein said apoptosis is p53-independent apoptosis.
 17. (Previously Amended) The method according to claim 16 further comprising: administering or introducing a nucleic acid encoding Apoptin or a functional equivalent or fragment thereof, or Apoptin or a functional equivalent or fragment thereof.
 18. (Previously Amended) A pharmaceutical composition comprising a substance associated with Apoptin-associating proteinaceous substance, said substance comprising the nucleic acid of claim
 1. 19. (Previously Amended) The pharmaceutical composition of claim 18 further comprising: a second nucleic acid encoding Apoptin or a functional equivalent or fragment thereof or Apoptin or a functional equivalent or fragment thereof.
 20. (Previously Amended) The pharmaceutical composition of claim 18 wherein said substance is present in an amount sufficient to induce apoptosis.
 21. (Previously Amended) The pharmaceutical composition of claim 20 wherein said apoptosis is p53-independent.
 22. (Previously Amended) The pharmaceutical composition of claim 21, wherein the substance is present in an amount and manner for the treatment of a disease where enhanced cell proliferation or decreased cell death is observed.
 23. (Amended) The pharmaceutical composition of claim 22 wherein said disease comprises cancer or an autoimmune disease.
 24. (Twice Amended) A method for treating an individual carrying a disease where enhanced cell proliferation or decreased cell death is observed comprising treating said individual with the pharmaceutical composition of claim
 18. 25. (Twice Amended) A method for detecting a presence of cancer cells or cells that are cancer prone in a sample of cells comprising transfecting cells in said sample with the nucleic acid of claim 1 and determining a percentage of apoptosis of cells in said sample.
 26. (Twice Amended) A method for detecting a presence of cancer cells or cells that are cancer prone in a sample of cells comprising transfecting cells in said sample with the nucleic acid according to claim 1 and determining a intracellular localization of a protemaceous substance derived from said nucleic acid or a vector in said sample of said cells.
 27. (Amended) The method according to claim 26 wherein a presence of said proteinaceous substance in said cells is detected by immunostaining said cells with an antibody.
 29. (Twice Amended) A method for identifying a putative cancer-inducing agent, said method comprising: submitting a sample of cells to said agent, and detecting a presence of cancer cells or cells that are cancer prone in the sample of cells with the method according to claim
 25. 30. The method according to claim 29 wherein said putative cancer-inducing agent comprises a genome or functional fragment thereof. 